Nucleic acids encoding human inositolpolyphosphate 5-phosphatase

ABSTRACT

Reagents which regulate human inositol polyphosphate 5-phosphatase and reagents which bind to human inositol polyphosphate 5-phosphatase gene products can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including, but not limited to, COPD, asthma, diabetes, and cancer.

This application incorporates by reference Ser. No. 60/243,745 filedOct. 30, 2000, Ser. No. 60/257,302 filed Dec. 26, 2000 and Ser. No.60/314,660 filed Aug. 27, 2001.

TECHNICAL FIELD OF THE INVENTION

The invention relates to the area of enzyme regulation. Moreparticularly, the invention relates to the regulation of human inositolpolyphosphate 5-phosphatase.

BACKGROUND OF TH INVENTION

SHC and GRB2 protein signaling molecules form a complex in response togrowth factor or oncogenic transformation, as described inRozakis-Adeock et al Nature 360: 689-92 (1992). U.S. Pat. No. 6,090,621.These proteins are thought to transmit mitogenic signals from receptorand non-receptor tyrosine kinases to ras, a member of a major class ofoncogenes and proto-oncogenes that encode G proteins that are located onthe inner face of the plasma membrane, where they bind and hydrolyzeGTP. Ras proteins are involved in an unknown way in growth-factorstimulation of cell proliferation, as described in Alberts et al.,MOLECULAR BIOLOGY OF THE CELL (second edition, Garland publishing NewYork, 1989) pp. 699 and 705. The precise mechanism of the action of rasremains unknown, as indicated in Lowenstein et al Cell 70: 431-42 (1992)and Gale et al Nature 363: 88-92 (1993).

By expression interaction cloning, the GRB2 SH3 domains were found tobind to a GRB2-associated signaling inositol polyphosphate 5-phosphatase(called SIP-110), a 110 kDa protein believed to be involved in signalingevents that follow growth factor stimulation, and occur between the cellsurface and transcriptional activation events. Furthermore, SIP-110 isbelieved to participate in modulating signaling by ras and byphosphatidyl inositol 3-kinase (PI 3-kinase), two known regulators ofcell growth. It would be advantageous in the process of elucidating themechanism of ras, PI 3-kinase and other signaling molecules andpathways, to discover other signaling molecules that participate in thesignal transduction that modulates the activity of the ras pathway, thePI 3-kinase pathway, the MAP kinase pathway, the calcium signalingpathway and other signaling pathways such that cellular responsesincluding growth and proliferation may be regulated by regulating suchsignaling molecules.

Activation of phosphatidylinositol 3′-kinase (PI 3-kinase) by growthfactors and oncogenes has been implicated as a critical step inmitogenic signaling and cellular transformation, as described in Cantleyet al, Cell 64: 281-302 (1991), Kapeller and Cantley. Bioessays 16:565-76 (1994), and Stephens et al, Biochim Biophys Acta 1179: 27-75(1993), PI 3-kinase consists of 85 kDa and 110 kDa subunits whichassociate with receptor tyrosine kinases and intracellular signalingmolecules in response to treatment with growth factors or in transformedcells. Blockade of PI 3-kinase function either by mutagenesis or withpharmacological inhibitors prevents mitogenic signaling. Further, twoproducts of PI 3-kinase, PtdIns(3,4,5)P₃ and PtdIns=(3,4)P₂, increase incells treated with mitogenic stimuli as Hawkins, et al. Nature 358:157-910, (1992) and Klippel et al, Molecular and Cellular Biology 16:4117-4127 (1996). The products of PI 3-kase are presumed to act assecond messengers or as regulators of protein-protein interactions. Theregulation of PI 3-kinase activity during signaling is less wellstudied.

Changes in subcellular localization, in phosphorylation state and inconformation of the enzyme have been suggested to contribute toactivation but little is known about how PI 3-kinase might bedown-regulated. It would be advantageous to discover and characterizemolecules implicated in PI 3-kinase mediated pathways, as a means tolearning how to regulate PI 3-kinase. A number of distinct forms ofinositol polyphosphate 5-phosphatase have been identified. Erneaux etal., Biochim. Biophys. Acta 1436, 185-99, 1998. There is, therefore, thepossibility that additional forms of this enzyme exist which can beregulated to provide therapeutic effects.

SUMMARY OF THE INVENTION

It is an object of the invention to provide reagents and methods ofregulating a human inositol polyphosphate 5-phosphatase. This and otherobjects of the invention are provided by one or more of the embodimentsdescribed below.

One embodiment of the invention is a inositol polyphosphate5-phosphatase polypeptide comprising an amino acid sequence selectedfrom the group consisting of:

-   amino acid sequences which are at least about 85% identical to the    amino acid sequence shown in SEQ ID NO: 2;-   the amino acid sequence shown in SEQ ID NO: 2; amino acid sequences    which are at least about 85% identical to the amino acid sequence    shown in SEQ ID NO: 12; and-   the amino acid sequence shown in SEQ ID NO: 12.

Yet another embodiment of the invention is a method of screening foragents which decrease extracellular matrix degradation. A test compoundis contacted with a inositol polyphosphate 5-phosphatase polypeptidecomprising an amino acid sequence selected from the group consisting of:

-   amino acid sequences which are at least about 85% identical to the    amino acid sequence shown in SEQ ID NO: 2;-   the amino acid sequence shown in SEQ ID NO: 2;-   amino acid sequences which are at least about 85% identical to the    amino acid sequence shown in SEQ ID NO: 12; and-   the amino acid sequence shown in SEQ ID NO: 12.

Binding between the test compound and the inositol polyphosphate5-phosphatase polypeptide is detected. A test compound which binds tothe inositol polyphosphate 5-phosphatase polypeptide is therebyidentified as a potential agent for decreasing extracellular matrixdegradation. The agent can work by decreasing the activity of theinositol polyphosphate 5-phosphatase.

Another embodiment of the invention is a method of screening for agentswhich decrease extracellular matrix degradation. A test compound iscontacted with a polynucleotide encoding a inositol polyphosphate5-phosphatase polypeptide, wherein the polynucleotide comprises anucleotide sequence selected from the group consisting of:

-   nucleotide sequences which are at least about 50% identical to the    nucleotide sequence shown in SEQ ID NO: 1;-   the nucleotide sequence shown in SEQ ID NO: 1;-   nucleotide sequences which are at least about 50% identical to the    nucleotide sequence shown in SEQ ID NO: 11; and-   the nucleotide sequence shown in SEQ ID NO: 11.

Binding of the test compound to the polynucleotide is detected. A testcompound which binds to the polynucleotide is identified as a potentialagent for decreasing extracellular matrix degradation. The agent canwork by decreasing the amount of the inositol polyphosphate5-phosphatase through interacting with the inositol polyphosphate5-phosphatase mRNA.

Another embodiment of the invention is a method of screening for agentswhich regulate extracellular matrix degradation. A test compound iscontacted with a inositol polyphosphate 5-phosphatase polypeptidecomprising an amino acid sequence selected from the group consisting of:

-   amino acid sequences which are at least about 85% identical to the    amino acid sequence shown in SEQ ID NO: 2;-   the amino acid sequence shown in SEQ ID NO: 2;-   amino acid sequences which are at least about 85% identical to the    amino acid sequence shown in SEQ ID NO: 12; and-   the amino acid sequence shown in SEQ ID NO:12.

A inositol polyphosphate 5-phosphatase activity of the polypeptide isdetected. A test compound which increases inositol polyphosphate5-phosphatase activity of the polypeptide relative to inositolpolyphosphate 5-phosphatase activity in the absence of the test compoundis thereby identified as a potential agent for increasing extracellularmatrix degradation. A test compound which decreases inositolpolyphosphate 5-phosphatase activity of the polypeptide relative toinositol polyphosphate 5-phosphatase activity in the absence of the testcompound is thereby identified as a potential agent for decreasingextracellular matrix degradation.

Even another embodiment of the invention is a method of screening foragents which decrease extracellular matrix degradation. A test compoundis contacted with a inositol polyphosphate 5-phosphatase product of apolynucleotide which comprises a nucleotide sequence selected from thegroup consisting of:

-   nucleotide sequences which are at least about 50% identical to the    nucleotide sequence shown in SEQ ID NO: 1;-   the nucleotide sequence shown in SEQ ID NO: 1;-   nucleotide sequences which are at least about 50% identical to the    nucleotide sequence shown in SEQ ID NO: 11; and-   the nucleotide sequence shown in SEQ ID NO: 11.

Binding of the test compound to the inositol polyphosphate 5-phosphataseproduct is detected A test compound which binds to the inositolpolyphosphate 5-phosphatase product is thereby identified as a potentialagent for decreasing extracellular matrix degradation.

Still another embodiment of the invention is a method of reducingextracellular matrix degradation. A cell is contacted with a reagentwhich specifically binds to a polynucleotide encoding a inositolpolyphosphate 5-phosphatase polypeptide or the product encoded by thepolynucleotide, wherein the polynucleotide comprises a nucleotidesequence selected from the group consisting of:

-   nucleotide sequences which are at least about 50% identical to the    nucleotide sequence shown in SEQ ID NO: 1;-   the nucleotide sequence shown in SEQ ID NO: 1;-   nucleotide sequences which are at least about 50% identical to the    nucleotide sequence shown in SEQ ID NO: 11; and-   the nucleotide sequence shown in SEQ ID NO: 11.

Inositol polyphosphate 5-phosphatase activity in the cell is therebydecreased.

The invention thus provides a human inositol polyphosphate 5-phosphatasewhich can be used to identify test compounds which may act, for example,as activators or inhibitors at the enzyme's active site. Human inositolpolyphosphate 5-phosphatase and fragments thereof also are useful inraising specific antibodies which can block the enzyme and effectivelyreduce its activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the DNA-sequence encoding a inositol polyphosphate5-phosphatase Polypeptide (SEQ ID NO: 1).

FIG. 2 shows the amino acid sequence deduced from the DNA-sequence ofFIG. 1 (SEQ ID NO: 2).

FIG. 3 shows the amino acid sequence of the protein identified by tremb1Accession No. AB032551 (SEQ ID NO: 3).

FIG. 4 shows the DNA-sequence encoding a inositol polyphosphate5-phosphatase Polypeptide (SEQ ED NO: 4).

FIG. 5 shows the DNA-sequence encoding a inositol polyphosphate5-phosphatase Polypeptide (SEQ ID NO: 5).

FIG. 6 shows the DNA-sequence encoding a inositol polyphosphate5-phosphatase Polypeptide (SEQ ID NO: 6).

FIG. 7 shows the DNA-sequence encoding a inositol polyphosphate5-phosphatase Polypeptide (SEQ ID NO: 7).

FIG. 8 shows the DNA-sequence encoding a inositol polyphosphate5-phosphatase Polypeptide (SEQ ID NO:8).

FIG. 9 shows the DNA-sequence encoding a inositol polyphosphate5-phosphatase Polypeptide (SEQ ID NO: 9).

FIG. 10 shows the DNA-sequence encoding a inositol polyphosphate5-phosphatase Polypeptide (SEQ ID NO: 10).

FIG. 11 shows the DNA-sequence encoding a inositol polyphosphate5-phosphatase Polypeptide (SEQ ID NO: 11).

FIG. 12 shows the amino acid sequence deduced from the DNA-sequence ofFIG. 11 (SEQ ID NO: 12).

FIG. 13 shows the BLASTP alignment of human inositol polyphosphate5-phosphatase (SEQ ID NO: 2) with the protein identified with tremb1Accession No. AB032551 (SEQ ID NO: 3).

FIG. 14 shows the BLASTP—alignment of 249_genewise_pro (SEQ ID NO: 12)against tremb1|AB032551|AB032551_(—)1(SEQ ID NO: 3).

FIG. 15 shows the HMMPFAM—alignment of 249_genewise_pro (SEQ ID NO: 12)against pfam|hmm|IPPc (Inositol polyphosphate phosphatase family, c).

FIG. 16 shows the gene expression of human inositol polyphosphate5-phosphatase in human tissues relevant for diabetes and obesity asdetermined by RT-PCR with 35 cycles performed with gene specific primersaccording to a standard procedure as known in the art (described e.g. inEXAMPLE 6).

The following tissues are represented: Lane 1—Liver, lane 2—SkeletalMuscle, lane 3—Hypothalamus, lane 4—Islets, lane 5—Adipose Sub., lane6—Adipose Mes., lane 7—Genomic DNA, lane 8—No amplification conrtol and,lane 9—No template control. Expression is shown in lanes 2, 3 and 4.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to an isolated polynucleotide encoding a inositolpolyphosphate 5-phosphatase polypeptide and being selected from thegroup consisting of:

-   a) a polynucleotide encoding a inositol polyphosphate 5-phosphatase    polypeptide comprising an amino acid sequence selected from the    group consisting of:    -   amino acid sequences which are at least about 85% identical to    -   the amino acid sequence shown in SEQ ID NO: 2;    -   the amino acid sequence shown in SEQ ID NO: 2;    -   amino acid sequences which are at least about 85% identical to    -   the amino acid sequence shown in SEQ ID NO: 12; and    -   the amino acid sequence shown in SEQ ID NO: 12.-   b) a polynucleotide comprising the sequence of SEQ ID NOS: 1 or 11;-   c) a polynucleotide which hybridizes under stringent conditions to a    polynucleotide specified in (a) and (b);-   d) a polynucleotide the sequence of which deviates from the    polynucleotide sequences specified in (a) to (c) due to the    degeneration of the genetic code; and-   e) a polynucleotide which represents a fragment, derivative or    allelic variation of a polynucteotide sequence specified in (a) to    (d).

Furthermore, it has been discovered by the present applicant that anovel inositol polyphosphate 5-phosphatase, particularly a humaninositol polyphosphate 5-phosphatase, is a discovery of the presentinvention. Human inositol polyphosphate 5-phosphatase comprises theamino acid sequence shown in SEQ ID NOS: 2 AND 12. A coding sequence forhuman inositol polyphosphate 5-phosphatase is shown in SEQ ID NOS: 1 AND11. Related ESTs (SEQ ID NOS: 4-10) are expressed in head and neck,whole brain, infant brain, kidney, uterus, thyroid tumor, and mammarygland. Known inositol polyphosphate 5-phosphatases have been detected inlung, thymus, testes, placenta, heart, brain, kidney, ovary, and colon.Speed et al., Eur. J. Biochem. 234, 216-24, 1995; Kudo et al., BrainRes. Mol Brain Res. 75, 172-77, 2000. Inositol polyphosphate5-phosphatase also has been detected in membrane ruffles. Mochizuki &Takenawa, J. Biol. Chem. 274, 36790-95, 1999.

Human inositol polyphosphate 5-phosphatase is 84% identical over 621amino acids to the protein identified with tremb1 Accession No. AB032551and annotated as “proline-rich inositol polyphosphate 5-phosphatase”(FIG. 13).

Human inositol polyphosphate 5-phosphatase of the invention is expectedto be useful for the same purposes as previously identified inositolpolyphosphate 5-phosphatase enzymes. Human inositol polyphosphate5-phosphatase is believed to be useful in therapeutic methods to treatdisorders such as COPD, asthma, diabetes, and cancer. Human inositolpolyphosphate 5-phosphatase also can be used to screen for humaninositol polyphosphate 5-phosphatase activators and inhibitors.

Polypeptides

Human inositol polyphosphate 5-phosphatase polypeptides according to theinvention comprise at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150,175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750contiguous amino acids selected from the amino acid sequence shown inSEQ ID NOS:2 AND 12 or a biologically active variant thereof, as definedbelow. A inositol polyphosphate 5-phosphatase polypeptide of theinvention therefore can be a portion of a inositol polyphosphate5-phosphatase protein, a full-length inositol polyphosphate5-phosphatase protein, or a fusion protein comprising all or a portionof a inositol polyphosphate 5-phosphatase protein.

Biologically Active Variants

Human inositol polyphosphate 5-phosphatase polypeptide variants whichare biologically active, e.g., retain an inositol polyphosphate5-phosphatase activity, also are inositol polyphosphate 5-phosphatasepolypeptides. Preferably, naturally or non-naturally occurring inositolpolyphosphate 5-phosphatase polypeptide variants have amino acidsequences which are at least about 85, 90, 96, 96, or 98% identical tothe amino acid sequence shown in SEQ ID NOS: 2 AND 12 or a fragmentthereof. Percent identity between a putative inositol polyphosphate5-phosphatase poly-peptide variant and an amino acid sequence of SEQ IDNOS: 2 AND 12 is determined using the Blast2 alignment program(Blosum62, Expect 10, standard genetic codes).

Variations in percent identity can be due, for example, to amino acidsubstitutions, insertions, or deletions. Amino acid substitutions aredefined as one for one amino acid replacements. They are conservative innature when the substituted amino acid has similar structural and/orchemical properties. Examples of conservative replacements aresubstitution of a leucine with an isoleucine or valine, an aspartatewith a glutamate, or a threonine with a serine.

Amino acid insertions or deletions are changes to or within an aminoacid sequence. They typically fall in the range of about 1 to 5 aminoacids. Guidance in determining which amino acid residues can besubstituted, inserted, or deleted without abolishing biological orimmunological activity of a inositol polyphosphate 5-phosphatasepolypeptide can be found using computer programs well known in the art,such as DNASTAR software. Whether an amino acid change results in abiologically active inositol polyphosphate 5-phosphatase polypeptide canreadily be determined by assaying for inositol polyphosphate5-phosphatase activity, as described for example, in U.S. Pat. No.6,090,621.

Fusion Proteins

Fusion proteins are useful for generating antibodies against inositolpolyphosphate 5-phosphatase polypeptide amino acid sequences and for usein various assay systems. For example, fusion proteins can be used toidentify proteins which interact with portions of a inositolpolyphosphate 5-phosphatase polypeptide. Protein affinity chromatographyor library-based assays for protein-protein interactions, such as theyeast two-hybrid or phage display systems, can be used for this purpose.Such methods are well known in the art and also can be used as drugscreens.

A inositol polyphosphate 5-phosphatase polypeptide fusion proteincomprises two polypeptide segments fused together by means of a peptidebond. The first poly-peptide segment comprises at least 6, 10, 15, 20,25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, or 750 contiguous amino acids of SEQ ID NOS:2 AND 12 orof a biologically active variant, such as those described above. Thefirst polypeptide segment also can comprise full-length inositolpolyphosphate 5-phosphatase protein.

The second polypeptide segment can be a full-length protein or a proteinfragment. Proteins commonly used in fusion protein construction includeβ-galactosidase, β-glucuronidase, green fluorescent protein (GFP),autofluorescent proteins, including blue fluorescent protein (BFP),glutathione-S-transferase (GST), luciferase, horseradish peroxidase(HRP), and chloramphenicol acetyltransferase (CAT). Additionally,epitope tags are used in fusion protein constructions, includinghistidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myctags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructionscan include maltose binding protein (MBP), S-tag, Lex a DNA bindingdomain (DBD) fusions, GAL4 DNA binding domain fusions, and herpessimplex virus (HSV) BP16 protein fusions. A fusion protein also can beengineered to contain a cleavage site located between the inositolpolyphosphate 5-phosphatase polypeptide-encoding sequence and theheterologous protein sequence, so that the inositol polyphosphate5-phosphatase polypeptide can be cleaved and purified away from theheterologous moiety.

A fusion protein can be synthesized chemically, as is known in the art.Preferably, a fusion protein is produced by covalently linking twopolypeptide segments or by standard procedures in the art of molecularbiology. Recombinant DNA methods can be used to prepare fusion proteins,for example, by making a DNA construct which comprises coding sequencesselected from the complement of SEQ ID NOS: 1 AND 11 in proper readingframe with nucleotides encoding the second polypeptide segment andexpressing the DNA construct in a host cell, as is known in the art.Many kits for constructing fusion proteins are available from companiessuch as Promega Corporation (Madison, Wis.), Stratagene (La Jolla,Calif.), CLONTECH (Mountain View, Calif.), Santa Cruz BioTechnology(Santa Cruz, Calif.), MBL International Corporation (MIC; Watertown,Mass.), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).

Identification of Species Homologs

Species homologs of human inositol polyphosphate 5-phosphatasepolypeptide can be obtained using inositol polyphosphate 5-phosphatasepolypeptide polynucleotides (described below) to make suitable probes orprimers for screening cDNA expression libraries from other species, suchas mice, monkeys, or yeast, identifying cDNAs which encode homologs ofinositol polyphosphate 5-phosphatase polypeptide, and expressing thecDNAs as is known in the art.

Polynucleotides

A inositol polyphosphate 5-phosphatase polynucleotide can be single- ordouble-stranded and comprises a coding sequence or the complement of acoding sequence for a inositol polyphosphate 5-phosphatase polypeptide.A coding sequence for human inositol polyphosphate 5-phosphatase isshown in SEQ ID NOS: 1 AND 11.

Degenerate nucleotide sequences encoding human inositol polyphosphate5-phosphatase polypeptides, as well as homologous nucleotide sequenceswhich are at least about 50, 55, 60, 65, 70, preferably about 75, 90,96, or 98% identical to the nucleotide sequence shown in SEQ ID NOS: 1AND 11 or its complement also are inositol polyphosphate 5-phosphatasepolynucleotides. Percent sequence identity between the sequences of twopolynucleotides is determined using computer programs such as ALIGNwhich employ the FASTA algorithm, using an affine gap search with a gapopen penalty of −12 and a gap extension penalty of −2. Complementary DNA(cDNA) molecules, species homologs, and variants of inositolpolyphosphate 5-phosphatase polynucleotides which encode biologicallyactive inositol polyphosphate 5-phosphatase polypeptides also areinositol polyphosphate 5-phosphatase polynucleotides. Polynucleotidescomprising at least 6, 7, 8, 9, 10, 12, 15, 18, 20, or 25 contiguousnucleotides of SEQ ID NOS: 1 AND 11 or its complement also are humaninositol polyphosphate 5-phosphatase polynucleotides. Suchpolynucleotides can be used, for example, as hybridization probes orantisense oligonucleotides.

Identification of Polynucleotide Variants and Homologs

Variants and homologs of the inositol polyphosphate 5-phosphatasepolynucleotides described above also are inositol polyphosphate5-phosphatase polynucleotides. Typically, homologous inositolpolyphosphate 5-phosphatase polynucleotide sequences can be identifiedby hybridization of candidate polynucleotides to known inositolpolyphosphate 5-phosphatase polynucleotides under stringent conditions,as is known in the art. For example, using the following washconditions-2×SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS,room temperature twice, 30 minutes each; then 2×SSC, 0.1% SDS, 50° C.once, 30 minutes; then 2×SSC, room temperature twice, 10 minuteseach—homologous sequences can be identified which contain at most about25-30% basepair mismatches. More preferably, homologous nucleic acidstrands contain 15-25% basepair mismatches, even more preferably 5-15%basepair mismatches.

Species homologs of the inositol polyphosphate 5-phosphatasepolynucleotides disclosed herein also can be identified by makingsuitable probes or primers and screening cDNA expression libraries fromother species, such as mice, monkeys, or yeast. Human variants ofinositol polyphosphate 5-phosphatase polynucleotides can be identified,for example, by screening human cDNA expression libraries. It is wellknown that the T_(m) of a double stranded DNA decreases by l-1.5° C.with every 1% decrease in homology (Bonner et al., J. Mol. Biol. 81, 123(1973). Variants of human inositol polyphosphate 5-phosphatasepolynucleotides or inositol polyphosphate 5-phosphatase polynucleotidesof other species can therefore be identified by hybridizing a putativehomologous inositol polyphosphate 5-phosphatase polynucleotide with apolynucleotide having a nucleotide sequence of SEQ ID NOS: 1 AND 11 orthe complement thereof to form a test hybrid. The melting temperature ofthe test hybrid is compared with the melting temperature of a hybridcomprising polynucleotides having perfectly complementary nucleotidesequences, and the number or percent of basepair mismatches within thetest hybrid is calculated.

Nucleotide sequences which hybridize to inositol polyphosphate5-phosphatase polynucleotides or their complements following stringenthybridization and/or wash conditions also are inositol polyphosphate5-phosphatase polynucleotides. Stringent wash conditions are well knownand understood in the art and are disclosed, for example, in Sambrook etal., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages9.50-9.51.

Typically, for stringent hybridization conditions a combination oftemperature and salt concentration should be chosen that isapproximately 12-20° C. below the calculated T_(m) of the hybrid understudy. The T_(m) of a hybrid between a inositol polyphosphate5-phosphatase polynucleotide having a nucleotide sequence shown in SEQID NOS:1 AND 11 or the complement thereof and a polynucleotide sequencewhich is at least about 50, preferably about 75, 90, 96, or 98%identical to one of those nucleotide sequences can be calculated, forexample, using the equation of Bolton and McCarthy, Proc. Natl. Acad.Sci. U.S.A. 48, 1390 (1962):

-   -   T_(m)=81.5° C.−16.6(log10[Na⁺]+0.41(% G+C)−0.63(%        formamide)−600/l), where l=the length of the hybrid in        basepairs.

Stringent wash conditions include, for example, 4×SSC at 65° C., or 50%formamide, 4×SSC at 42° C., or 0.5×SSC, 0.1% SDS at 65° C. Highlystringent wash conditions include, for example, 0.2×SSC at 65° C.

Preparation of Polynucleotides

A inositol polyphosphate 5-phosphatase polynucleotide can be isolatedfree of other cellular components such as membrane components, proteins,and lipids. Polynucleotides can be made by a cell and isolated usingstandard nucleic acid purification techniques, or synthesized using anamplification technique, such as the polymerase chain reaction (PCR), orby using an automatic synthesizer. Methods for isolating polynucleotidesare routine and are known in the art. Any such technique for obtaining apolynucleotide can be used to obtain isolated inositol polyphosphate5-phosphatase polynucleotides. For example, restriction enzymes andprobes can be used to isolate polynucleotide fragments which comprisesinositol polyphosphate 5-phosphatase nucleotide sequences. Isolatedpolynucleotides are in preparations which are free or at least 70, 80,or 90% free of other molecules.

Human inositol polyphosphate 5-phosphatase cDNA molecules can be madewith standard molecular biology techniques, using inositol polyphosphate5-phosphatase mRNA as a template. Human inositol polyphosphate5-phosphatase cDNA molecules can thereafter be replicated usingmolecular biology techniques known in the art and disclosed in manualssuch as Sambrook et al. (1989). An amplification technique, such as PCR,can be used to obtain additional copies of polynucleotides of theinvention, using either human genomic DNA or cDNA as a template.

Alternatively, synthetic chemistry techniques can be used to synthesizesinositol polyphosphate 5-phosphatase polynucleotides. The degeneracy ofthe genetic code allows alternate nucleotide sequences to be synthesizedwhich will encode a inositol polyphosphate 5-phosphatase polypeptidehaving, for example, an amino acid sequence shown in SEQ ID NOS: 1 AND11 or a biologically active variant thereof.

Extending Polynucleotides

Various PCR-based methods can be used to extend the nucleic acidsequences disclosed herein to detect upstream sequences such aspromoters and regulatory elements. For example, restriction-site PCRuses universal primers to retrieve unknown sequence adjacent to a knownlocus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA isfirst amplified in the presence of a primer to a linker sequence and aprimer specific to the known region. The amplified sequences are thensubjected to a second round of PCR with the same linker primer andanother specific primer internal to the first one. Products of eachround of PCR are transcribed with an appropriate RNA polymerase andsequenced using reverse transcriptase.

Inverse PCR also can be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia et al., Nucleic AcidsRes. 16, 8186, 1988). Primers can be designed using commerciallyavailable software, such as OLIGO 4.06 Primer Analysis software(National Biosciences Inc., Plymouth, Minn.), to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

Another method which can be used is capture PCR, which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom et al., PCR Methods Applic.1, 111-119, 1991). In this method, multiple restriction enzymedigestions and ligations also can be used to place an engineereddouble-standed sequence into an unknown fragment of the DNA moleculebefore performing PCR.

Another method which can be used to retrieve unknown sequences is thatof Parker et al., Nucleic Acids Res. 19, 3055-3060, 1991). Additionally,PCR, nested primers, and PROMOTERFINDER libraries (CLONTECH, Palo Alto,Calif.) can be used to walk genomic DNA (CLONTECH, Palo Alto, Calif.).This process avoids the need to screen libraries and is useful infinding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Randomly-primedlibraries are preferable, in that they will contain more sequences whichcontain the 5′ regions of genes. Use of a randomly primed library may beespecially preferable for situations in which an oligo d(T) library doesnot yield a full-length cDNA. Genomic libraries can be useful forextension of sequence into 5′ non-transcribed regulatory regions.

Commercially available capillary electrophoresis systems can be used toanalyze the size or confirm the nucleotide sequence of PCR or sequencingproducts. For example, capillary sequencing can employ flowable polymersfor electrophoretic separation, four different fluorescent dyes (one foreach nucleotide) which are laser activated, and detection of the emittedwavelengths by a charge coupled device camera. Output/light intensitycan be converted to electrical signal using appropriate software (e.g.GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire processfrom loading of samples to computer analysis and electronic data displaycan be computer controlled. Capillary electrophoresis is especiallypreferable for the sequencing of small pieces of DNA which might bepresent in limited amounts in a particular sample.

Obtaining Polypeptides

Human inositol polyphosphate 5-phosphatase polypeptides can be obtained,for example, by purification from human cells, by expression of inositolpolyphosphate 5-phosphatase polynucleotides, or by direct chemicalsynthesis.

Protein Purification

Human inositol polyphosphate 5-phosphatase polypeptides can be purifiedfrom any cell which expresses the enzyme, including host cells whichhave been transfected with inositol polyphosphate 5-phosphataseexpression constructs. A purified inositol polyphosphate 5-phosphatasepolypeptide is separated from other compounds which normally associatewith the inositol polyphosphate 5-phosphatase polypeptide in the cell,such as certain proteins, carbohydrates, or lipids, using methodswell-known in the art. Such methods include, but are not limited to,size exclusion chromatography, ammonium sulfate fractionation, ionexchange chromatography, affinity chromatography, and preparative gelelectrophoresis. A preparation of purified inositol polyphosphate5-phosphatase polypeptides is at least 80% pure; preferably, thepreparations are 90%, 95%, or 99% pure. Purity of the preparations canbe assessed by any means known in the art, such as SDS-polyacrylamidegel electrophoresis.

Expression of Polynucleotides

To express a inositol polyphosphate 5-phosphatase polynucleotide, thepolynucleotide can be inserted into an expression vector which containsthe necessary elements for the transcription and translation of theinserted coding sequence. Methods which are well known to those skilledin the art can be used to construct expression vectors containingsequences encoding inositol polyphosphate 5-phosphatase polypeptides andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed, for example, in Sambrook et al. (1989) and in Ausubel et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York,N.Y., 1989.

A variety of expression vector/host systems can be utilized to containand express sequences encoding a inositol polyphosphate 5-phosphatasepolypeptide. These include, but are not limited to, microorganisms, suchas bacteria transformed with recombinant bacteriophage, plasmid, orcosmid DNA expression vectors; yeast transformed with yeast expressionvectors, insect cell systems infected with virus expression vectors(e.g., baculovirus), plant cell systems transformed with virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322plasmids), or animal cell systems.

The control elements or regulatory sequences are those non-translatedregions of the vector—enhancers, promoters, 5′ and 3′ untranslatedregions—which interact with host cellular proteins to carry outtranscription and translation. Such elements can vary in their strengthand specificity. Depending on the vector system and host utilized, anynumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, can be used. For example, whencloning in bacterial systems, inducible promoters such as the hybridlacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.)or pSPORT1 plasmid (Life Technologies) and the like can be used. Thebaculovirus polyhedrin promoter can be used in insect cells. Promotersor enhancers derived from the genomes of plant cells (e.g., heat shock,RUDISCO, and storage protein genes) or from plant viruses (e.g., viralpromoters or leader sequences) can be cloned into the vector. Inmammalian cell systems, promoters from mammalian genes or from mammalianviruses are preferable. If it is necessary to generate a cell line thatcontains multiple copies of a nucleotide sequence encoding a inositolpolyphosphate 5-phosphatase polypeptide, vectors based on SV40 or BBVcan be used with an appropriate selectable marker.

Bacterial and Yeast Expression Systems

In bacterial systems, a number of expression vectors can be selecteddepending upon the use intended for the inositol polyphosphate5-phosphatase polypeptide. For example, when a large quantity of ainositol polyphosphate 5-phosphatase polypeptide is needed for theinduction of antibodies, vectors which direct high level expression offusion proteins that are readily purified can be used. Such vectorsinclude, but are not limited to, multifunctional E. coli cloning andexpression vectors such as BLUESCRIPT (Stratagene). In a BLUESCRIPTvector, a sequence encoding the inositol polyphosphate 5-phosphatasepolypeptide can be ligated into the vector in frame with sequences forthe amino-terminal Met and the subsequent 7 residues of β-galactosidaseso that a hybrid protein is produced. pIN vectors (Van Heeke & Schuster,J. Biol. Chem. 264, 5503-5509, 1989) or pGEX vectors (Pomega, Madison,Wis.) also can be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems can bedesigned to include heparin, thrombin, or factor Xa protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

In the yeast Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH can be used. For reviews, see Ausubel et al. (1989) andGrant et al., Methods Enzymol. 153, 516-544, 1987.

Plant and Insect Expression Systems

If plant expression vectors are used, the expression of sequencesencoding inositol polyphosphate 5-phosphatase polypeptides can be drivenby any of a number of promoters. For example, viral promoters such asthe ³⁵S and 19S promoters of CaMV can be used alone or in combinationwith the omega leader sequence from TMV (Takamatsu, EMBO J. 6, 307-311,1987). Alternatively, plant promoters such as the small subunit ofRUBISCO or heat shock promoters can be used (Coruzzi et al., EMBO J. 3,1671-1680, 1984; Broglie et al., Science 224, 838-843, 1984; Winter etal., Results Probl. Cell Differ. 17, 85-105, 1991). These constructs canbe introduced into plant cells by direct DNA transformation or bypathogen-mediated transfection. Such techniques are described in anumber of generally available reviews (e.g., Hobbs or Murray, in MCGRAWHILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N.Y.,pp. 191-196, 1992).

An insect system also can be used to express a inositol polyphosphate5-phosphatase polypeptide. For example, in one such system Autographacalifornica nuclear polyhedrosis virus (AcNPV) is used as a vector toexpress foreign genes in Spodoptera frugiperda cells or in Trichoplusialarvae. Sequences encoding inositol polyphosphate 5-phosphatasepolypeptides can be cloned into a non-essential region of the virus,such as the polyhedrin gene, and placed under control of the polyhedrinpromoter. Successful insertion of inositol polyphosphate 5-phosphatasepolypeptides will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses can thenbe used to infect S. frugiperda cells or Trichoplusia larvae in whichinositol polyphosphate 5-phosphatase polypeptides can be expressed(Engelhard et al., Proc. Nat. Acad. Sci. 91, 3224-3227, 1994).

Mammalian Expression Systems

A number of viral-based expression systems can be used to expressinositol polyphosphate 5-phosphatase polypeptides in mammalian hostcells. For example, if an adenovirus is used as an expression vector,sequences encoding inositol polyphosphate 5-phosphatase polypeptides canbe ligated into an adenovirus transcription/translation complexcomprising the late promoter and tripartite leader sequence. Insertionin a non-essential E1 or E3 region of the viral genome can be used toobtain a viable virus which is capable of expressing a inositolpolyphosphate 5-phosphatase polypeptide in infected host cells (Logan &Shenk, Proc. Natl. Acad. Sci. 81, 3655-3659, 1984). If desired,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,can be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) also can be used to deliver largerfragments of DNA than can be contained and expressed in a plasmid. HACsof 6M to 10 M are constructed and delivered to cells via conventionaldelivery methods (e.g., liposomes, polycationic amino polymers, orvesicles).

Specific initiation signals also can be used to achieve more efficienttranslation of sequences encoding inositol polyphosphate 5-phosphatasepolypeptides. Such signals include the ATG initiation codon and adjacentsequences. In cases where sequences encoding a inositol polyphosphate5-phosphatase polypeptide, its initiation codon, and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals (including the ATG initiationcodon) should be provided. The initiation codon should be in the correctreading frame to ensure translation of the entire insert. Exogenoustranslational elements and initiation codons can be of various origins,both natural and synthetic. The efficiency of expression can be enhancedby the inclusion of enhancers which are appropriate for the particularcell system which is used (see Scharf et al., Results Probl. CellDiffer. 20, 125-162, 1994).

Host Cells

A host cell strain can be chosen for its ability to modulate theexpression of the inserted sequences or to process the expressedinositol polyphosphate 5-phosphatase polypeptide in the desired fashion.Such modifications of the polypeptide include, but are not limited to,acetylation, carboxylation, glycosylation, phosphorylation, lipidation,and acylation. Post-translational processing which cleaves a “prepro”form of the polypeptide also can be used to facilitate correctinsertion, folding and/or function. Different host cells which havespecific cellular machinery and characteristic mechanisms forpost-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and W138),are available from the American Type Culture Collection (ATCC; 10801University Boulevard, Manassas, Va. 20110-2209) and can be chosen toensure the correct modification and processing of the foreign protein.

Stable expression is preferred for long-term, high-yield production ofrecombinant proteins. For example, cell lines which stably expressinositol polyphosphate 5-phosphatase polypeptides can be transformedusing expression vectors which can contain viral origins of replicationand/or endogenous expression elements and a selectable marker gene onthe same or on a separate vector. Following the introduction of thevector, cells can be allowed to grow for 1-2 days in an enriched mediumbefore they are switched to a selective medium. The purpose of theselectable marker is to confer resistance to selection, and its presenceallows growth and recovery of cells which successfully express theintroduced inositol polyphosphate 5-phosphatase sequences. Resistantclones of stably transformed cells can be proliferated using tissueculture techniques appropriate to the cell type. See, for example,ANIMAL CELL CULTURE, R. I. Freshney, ed., 1986.

Any number of selection systems can be used to recover transformed celllines.

These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler et al., Cell 11, 223-32, 1977) and adeninephosphoribosyltransferase (Lowy et al., Cell 22, 817-23, 1980) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate (Wigler et al., Proc. Natl. Acad. Sci. 77, 3567-70, 1980),npt confers resistance to the aminoglycosides, neomycin and G418(Colbere-Garapin et al., J. Mol. Biol. 150, 1-14, 1981), and als and patconfer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murray, 1992, supra). Additionalselectable genes have been described. For example, trpB allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl.Acad. Sci. 85, 8047-51, 1988). Visible markers such as anthocyanins,β-glucuronidase and its substrate GUS, and luciferase and its substrateluciferin, can be used to identify transformants and to quantify theamount of transient or stable protein expression attributable to aspecific vector system (Rhodes et al., Methods Mol. Biol. 55, 121-131,1995).

Detecting Expression

Although the presence of marker gene expression suggests that theinositol polyphosphate 5-phosphatase polynucleotide is also present, itspresence and expression may need to be confirmed. For example, if asequence encoding a inositol polyphosphate 5-phosphatase polypeptide isinserted within a marker gene sequence, transformed cells containingsequences which encode a inositol polyphosphate 5-phosphatasepolypeptide can be identified by the absence of marker gene function.Alternatively, a marker gene can be placed in tandem with a sequenceencoding a inositol polyphosphate 5-phosphatase polypeptide under thecontrol of a single promoter. Expression of the marker gene in responseto induction or selection usually indicates expression of the inositolpolyphosphate 5-phosphatase polynucleotide.

Alternatively, host cells which contain a inositol polyphosphate5-phosphatase polynucleotide and which express a inositol polyphosphate5-phosphatase polypeptide can be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassayor immunoassay techniques which include membrane, solution, orchip-based technologies for the detection and/or quantification ofnucleic acid or protein. For example, the presence of a polynucleotidesequence encoding a inositol polyphosphate 5-phosphatase polypeptide canbe detected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or fragments or fragments of polynucleotides encoding a inositolpolyphosphate 5-phosphatase polypeptide. Nucleic acidamplification-based assays involve the use of oligonucleotides selectedfrom sequences encoding a inositol polyphosphate 5-phosphatasepolypeptide to detect transformants which contain a inositolpolyphosphate 5-phosphatase polynucleotide.

A variety of protocols for detecting and measuring the expression of ainositol polyphosphate 5-phosphatase polypeptide, using eitherpolyclonal or monoclonal antibodies specific for the polypeptide, areknown in the art. Examples include enzyme-linked immunosorbent assay(ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting(FACS). A two-site, monoclonal-based immunoassay using monoclonalantibodies reactive to two non-interfering epitopes on a inositolpolyphosphate 5-phosphatase polypeptide can be used, or a competitivebinding assay can be employed. These and other assays are described inHampton et al., SEROLOGICAL METHODS: A LABORATORY MANUAL, APS Press, St.Paul, Minn., 1990) and Maddox et al., J. Exp. Med. 158, 1211-1216,1983).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and can be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding inositolpolyphosphate 5-phosphatase polypeptides include oligolabeling, nicktranslation, end-labeling, or PCR amplification using a labelednucleotide. Alternatively, sequences encoding a inositol polyphosphate5-phosphatase polypeptide can be cloned into a vector for the productionof an mRNA probe. Such vectors are known in the art, are commerciallyavailable, and can be used to synthesize RNA probes in vitro by additionof labeled nucleotides and an appropriate RNA polymerase such as T7, T3,or SP6. These procedures can be conducted using a variety ofcommercially available kits (Amersham Pharmacia Biotech, Promega, and USBiochemical). Suitable reporter molecules or labels which can be usedfor ease of detection include radionuclides, enzymes, and fluorescent,chemiluminescent, or chromogenic agents, as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

Expression and Purification of Polypeptides

Host cells transformed with nucleotide sequences encoding a inositolpolyphosphate 5-phosphatase polypeptide can be cultured under conditionssuitable for the expression and recovery of the protein from cellculture. The polypeptide produced by a transformed cell can be secretedor contained intracellularly depending on the sequence and/or the vectorused. As will be understood by those of skill in the art, expressionvectors containing polynucleotides which encode inositol polyphosphate5-phosphatase polypeptides can be designed to contain signal sequenceswhich direct secretion of soluble inositol polyphosphate 5-phosphatasepolypeptides through a prokaryotic or eukaryotic cell membrane or whichdirect the membrane insertion of membrane-bound inositol polyphosphate5-phosphatase polypeptide.

As discussed above, other constructions can be used to join a sequenceencoding a inositol polyphosphate 5-phosphatase polypeptide to anucleotide sequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). Inclusion ofcleavable linker sequences such as those specific for Factor Xa orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and the inositol polyphosphate 5-phosphatase polypeptide also canbe used to facilitate purification. One such expression vector providesfor expression of a fusion protein containing a inositol polyphosphate5-phosphatase polypeptide and 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification by IMAC (immobilized metal ion affinitychromatography, as described in Porath et al., Prot. Exp. Purif. 3,263-281, 1992), while the enterokinase cleavage site provides a meansfor purifying the inositol polyphosphate 5-phosphatase polypeptide fromthe fusion protein. Vectors which contain fusion proteins are disclosedin Kroll et al., DNA Cell Biol. 12,441-453, 1993.

Chemical Synthesis

Sequences encoding a inositol polyphosphate 5-phosphatase polypeptidecan be synthesized, in whole or in part, using chemical methods wellknown in the art (see Caruthers et al., Nucl. Acids Res. Symp. Ser.215-223, 1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225-232, 1980).Alternatively, a inositol polyphosphate 5-phosphatase polypeptide itselfcan be produced using chemical methods to synthesize its amino acidsequence, such as by direct peptide synthesis using solid-phasetechniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Robergeet al., Science 269, 202-204, 1995). Protein synthesis can be performedusing manual techniques or by automation. Automated synthesis can beachieved, for example, using Applied Biosystems 431A Peptide Synthesizer(Peridn Elmer). Optionally, fragments of inositol polyphosphate5-phosphatase polypeptides can be separately synthesized and combinedusing chemical methods to produce a full-length molecule.

The newly synthesized peptide can be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton,PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., NewYork, N.Y., 1983). The composition of a synthetic inositol polyphosphate5-phosphatase polypeptide can be confirmed by amino acid analysis orsequencing (e.g., the Edman degradation procedure; see Creighton,supra). Additionally, any portion of the amino acid sequence of theinositol polyphosphate 5-phosphatase polypeptide can be altered duringdirect synthesis and/or combined using chemical methods with sequencesfrom other proteins to produce a variant polypeptide or a fusionprotein.

Production of Altered Polypeptides

As will be understood by those of skill in the art, it may beadvantageous to produce inositol polyphosphate 5-phosphatasepolypeptide-encoding nucleotide sequences possessing non-naturallyoccurring codons. For example, codons preferred by a particularprokaryotic or eukaryotic host can be selected to increase the rate ofprotein expression or to produce an RNA transcript having desirableproperties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

The nucleotide sequences disclosed herein can be engineered usingmethods generally known in the art to alter inositol polyphosphate5-phosphatase polypeptide-encoding sequences for a variety of reasons,including but not limited to, alterations which modify the cloning,processing, and/or expression of the polypeptide or mRNA product. DNAshuffling by random fragmentation and PCR reassembly of gene fragmentsand synthetic oligonucleotides can be used to engineer the nucleotidesequences. For example, site-directed mutagenesis can be used to insertnew restriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, introduce mutations, and so forth.

Antibodies

Any type of antibody known in the art can be generated to bindspecifically to an epitope of a inositol polyphosphate 5-phosphatasepolypeptide. “Antibody” as used herein includes intact immunoglobulinmolecules, as well as fragments thereon such as Fab, F(ab′)₂, and Fv,which are capable of binding an epitope of a inositol polyphosphate5-phosphatase polypeptide. Typically, at least 6, 8, 10, or 12contiguous amino acids are required to form an epitope. However,epitopes which involve non-contiguous amino acids may require more, e.g.at least 15, 25, or 50 amino acids.

An antibody which specifically binds to an epitope of a inositolpolyphosphate 5-phosphatase polypeptide can be used therapeutically, aswell as in immunochemical assays, such as Western blots, ELISAs,radioimmunoassays, immunohistochemical assays, immunoprecipitations, orother immunochemical assays known in the art. Various immunoassays canbe used to identify antibodies having the desired specificity. Numerousprotocols for competitive binding or immunoradiometric assays are wellknown in the art. Such immunoassays typically involve the measurement ofcomplex formation between an immunogen and an antibody whichspecifically binds to the immunogen.

Typically, an antibody which specifically binds to a inositolpolyphosphate 5-phosphatase polypeptide provides a detection signal atleast 5-, 10-, or 20-fold higher than a detection signal provided withother proteins when used in an immunochemical assay. Preferably,antibodies which specifically bind to inositol polyphosphate5-phosphatase polypeptides do not detect other proteins inimmunochemical assays and can immunoprecipitate a inositol polyphosphate5-phosphatase polypeptide from solution.

Human inositol polyphosphate 5-phosphatase polypeptides can be used toimmunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, orhuman, to produce polyclonal antibodies. If desired, a inositolpolyphosphate 5-phosphatase poly-peptide can be conjugated to a carrierprotein, such as bovine serum albumin, thyroglobulin, and keyhole limpethemocyanin. Depending on the host species, various adjuvants can be usedto increase the immunological response. Such adjuvants include, but arenot limited to, Freund's adjuvant, mineral gels (e.g., aluminumhydroxide), and surface active substances (e.g. lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol). Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially useful.

Monoclonal antibodies which specifically bind to a inositolpolyphosphate 5-phosphatase polypeptide can be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These techniques include, but are notlimited to, the hybridoma technique, the human B-cell hybridomatechnique, and the EBV-hybridoma technique (Kohler et al., Nature 256,495-497, 1985; Kozbor et al., J. Immunol. Methods 81, 31-42, 1985; Coteet al., Proc. Natl. Acad. Sc. 80, 2026-2030, 1983; Cole et al., Mol.Cell Biol. 62, 109-120, 1984).

In addition, techniques developed for the production of “chimericantibodies,” the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity, can be used (Morrison et al., Proc. Natl. Acad.Sci. 81, 6851-6855, 1984; Neuberger et al., Nature 312, 604-608, 1984;Takeda et al., Nature 314, 452-454, 1985). Monoclonal and otherantibodies also can be “humanized” to prevent a patient from mounting animmune response against the antibody when it is used therapeutically.Such antibodies may be sufficiently similar in sequence to humanantibodies to be used directly in therapy or may require alteration of afew key residues. Sequence differences between rodent antibodies andhuman sequences can be minimized by replacing residues which differ fromthose in the human sequences by site directed mutagenesis of individualresidues or by grating of entire complementarity determining regions.Alternatively, humanized antibodies can be produced using recombinantmethods, as described in GB2188638B. Antibodies which specifically bindto a inositol polyphosphate 5-phosphatase polypeptide can containantigen binding sites which are either partially or fully humanized, asdisclosed in U.S. Pat. No. 5,565,332.

Alternatively, techniques described for the production of single chainantibodies can be adapted using methods known in the art to producesingle chain antibodies which specifically bind to inositolpolyphosphate 5-phosphatase polypeptides. Antibodies with relatedspecificity, but of distinct idiotypic composition, can be generated bychain shuffling from random combinatorial immunoglobin libraries(Burton, Proc. Natl. Acad. Sci. 88, 11120-23, 1991).

Single-chain antibodies also can be constructed using a DNAamplification method, such as PCR, using hybridoma cDNA as a template(Thirion et al., 1996, Eur. J. Cancer Prev. 5, 507-11). Single-chainantibodies can be mono- or bispecific, and can be bivalent ortetravalent. Construction of tetravalent, bispecific single-chainantibodies is taught, for example, in Coloma & Morrison, 1997, Nat.Biotechnol. 15, 159-63. Construction of bivalent, bispecificsingle-chain antibodies is taught in Mallender & Voss, 1994, J. Biol.Chem. 269, 199-206.

A nucleotide sequence encoding a single-chain antibody can beconstructed using manual or automated nucleotide synthesis, cloned intoan expression construct using standard recombinant DNA methods, andintroduced into a cell to express the coding sequence, as describedbelow. Alternatively, single-chain antibodies can be produced directlyusing, for example, filamentous phage technology (Verhaar et al., 1995,Int. J. Cancer 61, 497-501; Nicholls et al., 1993, J. Immunol. Meth.165, 81-91).

Antibodies which specifically bind to inositol polyphosphate5-phosphatase poly-peptides also can be produced by inducing in vivoproduction in the lymphocyte population or by screening immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi et al., Proc. Natl. Acad. Sci. 86, 3833-3837,1989; Winter et al., Nature 349, 293-299, 1991).

Other types of antibodies can be constructed and used therapeutically inmethods of the invention. For example, chimeric antibodies can beconstructed as disclosed in WO 93/03151. Binding proteins which arederived from immunoglobulins and which are multivalent andmultispecific, such as the “diabodies” described in WO 94/13804, alsocan be prepared.

Antibodies according to the invention can be purified by methods wellknown in the art. For example, antibodies can be affinity purified bypassage over a column to which a inositol polyphosphate 5-phosphatasepolypeptide is bound. The bound antibodies can then be eluted from thecolumn using a buffer with a high salt concentration.

Antisense Oligonucleotides

Antisense oligonucleotides are nucleotide sequences which arecomplementary to a specific DNA or RNA sequence. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form complexes and block either transcription ortranslation. Preferably, an antisense oligonucleotide is at least 11nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40,45, or 50 or more nucleotides long. Longer sequences also can be used.Antisense oligonucleotide molecules can be provided in a DNA constructand introduced into a cell as described above to decrease the level ofinositol polyphosphate 5-phosphatase gene products in the cell.

Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides,or a combination of both. Oligonucleotides can be synthesized manuallyor by an automated synthesizer, by covalently linking the 5′ end of onenucleotide with the 3′ end of another nucleotide with non-phosphodiesterinternucleotide linkages such alkylphosphonates, phosphorothioates,phosphorodithioates, alkylphosphonothioates, alkylphosphonates,phosphoramidates, phosphate esters, carbamates, acetamidate,carboxymethyl esters, carbonates, and phosphate triesters. See Brown,Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72,1994; Uhlmann et al., Chem. Rev. 90, 543-583, 1990.

Modifications of inositol polyphosphate 5-phosphatase gene expressioncan be obtained by designing antisense oligonucleotides which will formduplexes to the control, 5′, or regulatory regions of the inositolpolyphosphate 5-phosphatase gene. Oligonucleotides derived from thetranscription initiation site, e.g., between positions −10 and +10 fromthe start site, are preferred. Similarly, inhibition can be achievedusing “triple helix” base-pairing methodology. Triple helix pairing isuseful because it causes inhibition of the ability of the double helixto open sufficiently for the binding of polymerases, transcriptionfactors, or chaperons. Therapeutic advances using triplex DNA have beendescribed in the literature (e.g., Gee et al., in Huber & Carr,MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura Publishing Co., Mt. Kisco,N.Y., 1994). An antisense oligonucleotide also can be designed to blocktranslation of mRNA by preventing the transcript from binding toribosomes.

Precise complementarity is not required for successful complex formationbetween an antisense oligonucleotide and the complementary sequence of ainositol polyphosphate 5-phosphatase polynucleotide. Antisenseoligonucleotides which comprise, for example, 2, 3, 4, or 5 or morestretches of contiguous nucleotides which are precisely complementary toa inositol polyphosphate 5-phosphatase polynucleotide, each separated bya stretch of contiguous nucleotides which are not complementary toadjacent inositol polyphosphate 5-phosphatase nucleotides, can providesufficient targeting specificity for inositol polyphosphate5-phosphatase mRNA. Preferably, each stretch of complementary contiguousnucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.Non-complementary intervening sequences are preferably 1, 2, 3, or 4nucleotides in length. One skilled in the art can easily use thecalculated melting point of an antisense-sense pair to determine thedegree of mismatching which will be tolerated between a particularantisense oligonucleotide and a particular inositol polyphosphate5-phosphatase polynucleotide sequence.

Antisense oligonucleotides can be modified without affecting theirability to hybridize to a inositol polyphosphate 5-phosphatasepolynucleotide. These modifications can be internal or at one or bothends of the antisense molecule. For example, internucleoside phosphatelinkages can be modified by adding cholesteryl or diamine moieties withvarying numbers of carbon residues between the amino groups and terminalribose. Modified bases and/or sugars, such as arabinose instead ofribose, or a 3′,5′-substituted oligonucleotide in which the 3′ hydroxylgroup or the 5′ phosphate group are substituted, also can be employed ina modified antisense oligonucleotide. These modified oligonucleotidescan be prepared by methods well known in the art. See, e.g., Agrawal etal., Trends Biotechnol. 10, 152-158, 1992; Uhlmann et al., Chem. Rev.90, 543-584, 1990; Uhlmann et al., Tetrahedron. Lett. 215, 3539-3542,1987.

Ribozymes

Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech,Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568;1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture &Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used toinhibit gene function by cleaving an RNA sequence, as is known in theart (e.g., Haseloff et al., U.S. Pat. No. 5,641,673). The mechanism ofribozyme action involves sequence-specific hybridization of the ribozymemolecule to complementary target RNA, followed by endonucleolyticcleavage. Examples include engineered hammerhead motif ribozymemolecules that can specifically and efficiently catalyze endonucleolyticcleavage of specific nucleotide sequences.

The coding sequence of a inositol polyphosphate 5-phosphatasepolynucleotide can be used to generate ribozymes which will specificallybind to mRNA transcribed from the inositol polyphosphate 5-phosphatasepolynucleotide. Methods of designing and constructing ribozymes whichcan cleave other RNA molecules in trans in a highly sequence specificmanner have been developed and described in the art (see Haseloff et al.Nature 334, 585-591, 1988). For example, the cleavage activity ofribozymes can be targeted to specific RNAs by engineering a discrete“hybridization” region into the ribozyme. The hybridization regioncontains a sequence complementary to the target RNA and thusspecifically hybridizes with the target (see, for example, Gerlach etal., EP 321, 201).

Specific ribozyme cleavage sites within a inositol polyphosphate5-phosphatase RNA target can be identified by scanning the targetmolecule for ribozyme cleavage sites which include the followingsequences: GUA, GUU, and GUC. Once identified, short RNA sequences ofbetween 15 and 20 ribonucleotides corresponding to the region of thetarget RNA containing the cleavage site can be evaluated for secondarystructural features which may render the target inoperable. Suitabilityof candidate inositol polyphosphate 5-phosphatase RNA targets also canbe evaluated by testing accessibility to hybridization withcomplementary oligonucleotides using ribonuclease protection assays.Longer complementary sequences can be used to increase the affinity ofthe hybridization sequence for the target. The hybridizing and cleavageregions of the ribozyme can be integrally related such that uponhybridizing to the target RNA through the complementary regions, thecatalytic region of the ribozyme can cleave the target.

Ribozymes can be introduced into cells as part of a DNA construct.Mechanical methods, such as microinjection, liposome-mediatedtransfection, electroporation, or calcium phosphate precipitation, canbe used to introduce a ribozyme-containing DNA construct into cells inwhich it is desired to decrease inositol polyphosphate 5-phosphataseexpression. Alternatively, if it is desired that the cells stably retainthe DNA construct, the construct can be supplied on a plasmid andmaintained as a separate element or integrated into the genome of thecells, as is known in the art. A ribozyme-encoding DNA construct caninclude transcriptional regulatory elements, such as a promoter element,an enhancer or UAS element, and a transcriptional terminator signal, forcontrolling transcription of ribozymes in the cells.

As taught in Haseloff et al., U.S. Pat. No. 5,641,673, ribozymes can beengineered so that ribozyme expression will occur in response to factorswhich induce expression of a target gene. Ribozymes also can beengineered to provide an additional level of regulation, so thatdestruction of mRNA occurs only when both a ribozyme and a target geneare induced in the cells.

Differentially Expressed Genes

Described herein are methods for the identification of genes whoseproducts interact with human inositol polyphosphate 5-phosphatase. Suchgenes may represent genes which are differentially expressed indisorders including, but not limited to, cancer, COPD, diabetes, andasthma. Further, such genes may represent genes which are differentiallyregulated in response to manipulations relevant to the progression ortreatment of such diseases. Additionally, such genes may have atemporally modulated expression, increased or decreased at differentstages of tissue or organism development. A differentially expressedgene may also have its expression modulated under control versusexperimental conditions. In addition, the human inositol polyphosphate5-phosphatase gene or gene product may itself be tested for differentialexpression.

The degree to which expression differs in a normal versus a diseasedstate need only be large enough to be visualized via standardcharacterization techniques such as differential display techniques.Other such standard characterization techniques by which expressiondifferences may be visualized include but are not limited to,quantitative RT (reverse transcriptase), PCR, and Northern analysis.

Identification of Differentially Expressed Genes

To identify differentially expressed genes total RNA or, preferably,mRNA is isolated from tissues of interest. For example, RNA samples areobtained from tissues of experimental subjects and from correspondingtissues of control subjects. Any RNA isolation technique which does notselect against the isolation of mRNA may be utilized for thepurification of such RNA samples. See, for example, Ausubel et al., ed.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. NewYork, 1987-1993. Large numbers of tissue samples may readily beprocessed using techniques well known to those of skill in the art, suchas, for example, the single-step RNA isolation process of Chomczynski,U.S. Pat. No. 4,843,155.

Transcripts within the collected RNA samples which represent RNAproduced by differentially expressed genes are identified by methodswell known to those of skill in the art. They include, for example,differential screening (Tedder et al., Proc. Natl. Acad. Sci. U.S.A. 85,208-12, 1988), subtractive hybridization (Hedrick et al., Nature 308,149-53; Lee et al., Proc. Natl. Acad. Sci. USA. 88, 2825, 1984), and,preferably, differential display (Liang & Pardee, Science 257, 967-71,1992; U.S. Pat. No. 5,262,311).

The differential expression information may itself suggest relevantmethods for the treatment of disorders involving the human inositolpolyphosphate 5-phosphatase. For example, treatment may include amodulation of expression of the differentially expressed genes and/orthe gene encoding the human inositol polyphosphate 5-phosphatase. Thedifferential expression information may indicate whether the expressionor activity of the differentially expressed gene or gene product or thehuman inositol polyphosphate 5-phosphatase gene or gene product areup-regulated or down-regulated.

Screening Methods

The invention provides assays for screening test compounds which bind toor modulate the activity of a inositol polyphosphate 5-phosphatasepolypeptide or a inositol polyphosphate 5-phosphatase polynucleotide. Atest compound preferably binds to a inositol polyphosphate 5-phosphatasepolypeptide or polynucleotide. More preferably, a test compounddecreases or increases an inositol polyphosphate 5-phosphatase activityby at least about 10, preferably about 50, more preferably about 75, 90,or 100% relative to the absence of the test compound.

Test Compounds

Test compounds can be pharmacologic agents already known in the art orcan be compounds previously unknown to have any pharmacologicalactivity. The compounds can be naturally occurring or designed in thelaboratory. They can be isolated from microorganisms, animals, orplants, and can be produced recombinantly, or synthesized by chemicalmethods known in the art. If desired, test compounds can be obtainedusing any of the numerous combinatorial library methods known in theart, including but not limited to, biological libraries, spatiallyaddressable parallel solid phase or solution phase libraries, syntheticlibrary methods requiring deconvolution, the “one-bead one-compound”library method, and synthetic library methods using affinitychromatography selection. The biological library approach is limited topolypeptide libraries, while the other four approaches are applicable topolypeptide, non-peptide oligomer, or small molecule libraries ofcompounds. See Lam, Anticancer Drug Des. 12, 145, 1997.

Methods for the synthesis of molecular libraries are well known in theart (see, for example, DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90,6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994;Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2059,1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop etal., J. Med. Chem. 37, 1233, 1994). Libraries of compounds can bepresented in solution (see, e.g., Houghten, BioTechniques 13, 412-421,1992), or on beads (Lam, Nature 354, 82-84, 1991), chips (Fodor, Nature364, 555-556, 1993), bacteria or spores (Ladner, U.S. Pat. No.5,223,409), plasmids (Cull et al., Proc. Natl. Acad. Sci. USA. 89,1865-1869, 1992), or phage (Scott & Smith, Science 249, 386-390, 1990;Devlin, Science 249, 404-406, 1990); Cwirla et al., Proc. Natl. Acad.Sci. 97, 6378-6382, 1990; Felici, J. Mol. Biol. 222, 301-310, 1991; andLadner, U.S. Pat. No. 5,223,409).

High Throughput Screening

Test compounds can be screened for the ability to bind to inositolpolyphosphate 5-phosphatase polypeptides or polynucleotides or to affectinositol polyphosphate 5-phosphatase activity or inositol polyphosphate5-phosphatase gene expression using high throughput screening. Usinghigh throughput screening, many discrete compounds can be tested inparallel so that large numbers of test compounds can be quicklyscreened. The most widely established techniques utilize 96-wellmicrotiter plates. The wells of the microtiter plates typically requireassay volumes that range from 50 to 500 μl. In addition to the plates,many instruments, materials, pipettors, robotics, plate washers, andplate readers are commercially available to fit the 96-well format.

Alternatively, “free format assays,” or assays that have no physicalbarrier between samples, can be used. For example, an assay usingpigment cells (melanocytes) in a simple homogeneous assay forcombinatorial peptide libraries is described by Jayawickreme et al.,Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placedunder agarose in petri dishes, then beads that carry combinatorialcompounds are placed on the surface of the agarose. The combinatorialcompounds are partially released the compounds from the beads. Activecompounds can be visualized as dark pigment areas because, as thecompounds diffuse locally into the gel matrix, the active compoundscause the cells to change colors.

Another example of a free format assay is described by Chelsky,“Strategies for Screening Combinatorial Libraries: Novel and TraditionalApproaches,” reported at the First Annual Conference of The Society forBiomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995). Chelskyplaced a simple homogenous enzyme assay for carbonic anhydrase inside anagarose gel such that the enzyme in the gel would cause a color changethroughout the gel. Thereafter, beads carrying combinatorial compoundsvia a photolinker were placed inside the gel and the compounds werepartially released by UV-light Compounds that inhibited the enzyme wereobserved as local zones of inhibition having less color change.

Yet another example is described by Salmon et al., Molecular Diversity2, 57-63 (1996). In this example, combinatorial libraries were screenedfor compounds that had cytotoxic effects on cancer cells growing inagar.

Another high throughput screening method is described in Beutel et al.,U.S. Pat. No. 5,976,813. In this method, test samples are placed in aporous matrix. One or more assay components are then placed within, ontop of, or at the bottom of a matrix such as a gel, a plastic sheet, afilter, or other form of easily manipulated solid support. When samplesare introduced to the porous matrix they diffuse sufficiently slowly,such that the assays can be performed without the test samples runningtogether.

Binding Assays

For binding assays, the test compound is preferably a small moleculewhich binds to and occupies, for example, the active site of theinositol polyphosphate 5-phosphatase polypeptide, such that normalbiological activity is prevented. Examples of such small moleculesinclude, but are not limited to, small peptides or peptide-likemolecules.

In binding assays, either the test compound or the inositolpolyphosphate 5-phosphatase polypeptide can comprise a detectable label,such as a fluorescent, radioisotopic, chemiluminescent, or enzymaticlabel, such as horseradish peroxidase, alkaline phosphatase, orluciferase. Detection of a test compound which is bound to the inositolpolyphosphate 5-phosphatase polypeptide can then be accomplished, forexample, by direct counting of radioemmission, by scintillationcounting, or by determining conversion of an appropriate substrate to adetectable product.

Alternatively, binding of a test compound to a inositol polyphosphate5-phosphatase polypeptide can be determined without labeling either ofthe interactants. For example, a microphysiometer can be used to detectbinding of a test compound with a inositol polyphosphate 5-phosphatasepolypeptide. A microphysiometer (e.g., Cytosensor™) is an analyticalinstrument that measures the rate at which a cell acidifies itsenvironment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between a test compound and a inositol polyphosphate5-phosphatase polypeptide (McConnell et al., Science 257, 1906-1912,1992).

Determining the ability of a test compound to bind to a inositolpolyphosphate 5-phosphatase polypeptide also can be accomplished using atechnology such as real-time Bimolecular Interaction Analysis (BIA)(Sjolander & Urbaniczky, Anal. Chem. 63, 2338-2345, 1991, and Szabo etal., Curr. Opin. Struct. Biol. 5, 699-705, 1995). BIA is a technologyfor studying biospecific interactions in real time, without labeling anyof the interactants (e.g., BIAcore™). Changes in the optical phenomenonsurface plasmon resonance (SPR) can be used as an indication ofreal-time reactions between biological molecules.

In yet another aspect of the invention, a inositol polyphosphate5-phosphatase polypeptide can be used as a “bait protein” in atwo-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No.5,283,317; Zervos et al., Cell 72, 223-232, 1993; Madura et al., J.Biol. Chem. 268, 12046-12054, 1993; Bartel et al., BioTechniques 14,920-924, 1993; Iwabuchi et al., Oncogene 8, 1693-1696, 1993; and BrentW094/10300), to identify other proteins which bind to or interact withthe inositol polyphosphate 5-phosphatase polypeptide and modulate itsactivity.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. For example, in one construct, polynucleotide encoding ainositol polyphosphate 5-phosphatase polypeptide can be fused to apolynucleotide encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct a DNA sequence that encodesan unidentified protein (“prey” or “sample”) can be fused to apolynucleotide that codes for the activation domain of the knowntranscription factor. If the “bait” and the “prey” proteins are able tointeract in vivo to form an protein-dependent complex, the DNA-bindingand activation domains of the transcription factor are brought intoclose proximity. This proximity allows transcription of a reporter gene(e.g., LacZ), which is operably linked to a transcriptional regulatorysite responsive to the transcription factor. Expression of the reportergene can be detected, and cell colonies containing the functionaltranscription factor can be isolated and used to obtain the DNA sequenceencoding the protein which interacts with the inositol polyphosphate5-phosphatase polypeptide.

It may be desirable to immobilize either the inositol polyphosphate5-phosphatase polypeptide (or polynucleotide) or the test compound tofacilitate separation of bound from unbound forms of one or both of theinteractants, as well as to accommodate automation of the assay. Thus,either the inositol polyphosphate 5-phosphatase polypeptide (orpolynucleotide) or the test compound can be bound to a solid support.Suitable solid supports include, but are not limited to, glass orplastic slides, tissue culture plates, microtiter wells, tubes, siliconchips, or particles such as beads (including, but not limited to, latex,polystyrene, or glass beads). Any method known in the art can be used toattach the enzyme polypeptide (or polynucleotide) or test compound to asolid support, including use of covalent and non-covalent linkages,passive absorption, or pairs of binding moieties attached respectivelyto the polypeptide (or polynucleotide) or test compound and the solidsupport. Test compounds are preferably bound to the solid support in anarray, so that the location of individual test compounds can be tracked.Binding of a test compound to a inositol polyphosphate 5-phosphatasepolypeptide (or polynucleotide) can be accomplished in any vesselsuitable for containing the reactants. Examples of such vessels includemicrotiter plates, test tubes, and microcentrifuge tubes.

In one embodiment, the inositol polyphosphate 5-phosphatase polypeptideis a fusion protein comprising a domain that allows the inositolpolyphosphate 5-phosphatase polypeptide to be bound to a solid support.For example, glutathione-S-transferase fusion proteins can be adsorbedonto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and the non-adsorbed inositolpolyphosphate 5-phosphatase polypeptide; the mixture is then incubatedunder conditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components.Binding of the interactants can be determined either directly orindirectly, as described above. Alternatively, the complexes can bedissociated from the solid support before binding is determined.

Other techniques for immobilizing proteins or polynucleotides on a solidsupport also can be used in the screening assays of the invention. Forexample, either a inositol polyphosphate 5-phosphatase polypeptide (orpolynucleotide) or a test compound can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated inositolpolyphosphate 5-phosphatase polypeptides (or polynucleotides) or testcompounds can be prepared from biotin-NHS(N-hydroxysuccinimide) usingtechniques well known in the art (e.g., biotinylation idt, PierceChemicals, Rockford, Ill.) and immobilised in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies which specifically bind to a inositol polyphosphate5-phosphatase polypeptide, polynucleotide, or a test compound, but whichdo not interfere with a desired binding site, such as the active site ofthe inositol polyphosphate 5-phosphatase polypeptide, can be derivatizedto the wells of the plate. Unbound target or protein can be trapped inthe wells by antibody conjugation.

Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using ant-bodies which specifically bind to the inositolpolyphosphate 5-phosphatase polypeptide or test compound, enzyme-linkedassays which rely on detecting an activity of the inositol polyphosphate5-phosphatase polypeptide, and SDS gel electrophoresis undernon-reducing conditions.

Screening for test compounds which bind to a inositol polyphosphate5-phosphatase polypeptide or polynucleotide also can be carried out inan intact cell. Any cell which comprises a inositol polyphosphate5-phosphatase polypeptide or polynucleotide can be used in a cell-basedassay system. A inositol polyphosphate 5-phosphatase polynucleotide canbe naturally occurring in the cell or can be introduced using techniquessuch as those described above. Binding of the test compound to ainositol polyphosphate 5-phosphatase polypeptide or polynucleotide isdetermined as described above.

Enzyme Assays

Test compounds can be tested for the ability to increase or decrease theenzyme activity of a human inositol polyphosphate 5-phosphatasepolypeptide. Enzyme activity can be measured, for example, as describedin U.S. Pat. No. 6,090,621.

Enzyme assays can be carried out after contacting either a purifiedinositol polyphosphate 5-phosphatase polypeptide, a cell membranepreparation, or an intact cell with a test compound. A test compoundwhich decreases an enzyme activity of a inositol polyphosphate5-phosphatase polypeptide by at least about 10, preferably about 50,more preferably about 75, 90, or 100% is identified as a potentialtherapeutic agent for decreasing inositol polyphosphate 5-phosphataseactivity. A test compound which increases an enzyme activity of a humaninositol polyphosphate 5-phosphatase polypeptide by at least about 10,preferably about 50, more preferably about 75, 90, or 100% is identifiedas a potential therapeutic agent for increasing human inositolpolyphosphate 5-phosphatase activity.

Gene Expression

In another embodiment, test compounds which increase or decreaseinositol polyphosphate 5-phosphatase gene expression are identified. Ainositol polyphosphate 5-phosphatase polynucleotide is contacted with atest compound, and the expression of an RNA or polypeptide product ofthe inositol polyphosphate 5-phosphatase polynucleotide is determined.The level of expression of appropriate mRNA or polypeptide in thepresence of the test compound is compared to the level of expression ofmRNA or polypeptide in the absence of the test compound. The testcompound can then be identified as a modulator of expression based onthis comparison. For example, when expression of mRNA or polypeptide isgreater in the presence of the test compound than in its absence, thetest compound is identified as a stimulator or enhancer of the mRNA orpolypeptide expression. Alternatively, when expression of the mRNA orpolypeptide is less in the presence of the test compound than in itsabsence, the test compound is identified as an inhibitor of the mRNA orpolypeptide expression.

The level of inositol polyphosphate 5-phosphatase mRNA or polypeptideexpression in the cells can be determined by methods well known in theart for detecting mRNA or polypeptide. Either qualitative orquantitative methods can be used. The presence of polypeptide productsof a inositol polyphosphate 5-phosphatase polynucleotide can bedetermined, for example, using a variety of techniques known in the art,including immunochemical methods such as radioimmunoassay, Westernblotting, and immunohistochemistry. Alternatively, polypeptide synthesiscan be determined in vivo, in a cell culture, or in an in vitrotranslation system by detecting incorporation of labeled amino acidsinto a inositol polyphosphate 5-phosphatase polypeptide.

Such screening can be carried out either in a cell-free assay system orin an intact cell. Any cell which expresses a inositol polyphosphate5-phosphatase polynucleotide can be used in a cell-based assay system.The inositol polyphosphate 5-phosphatase polynucleotide can be naturallyoccurring in the cell or can be introduced using techniques such asthose described above. Either a primary culture or an established cellline, such as CHO or human embryonic kidney 293 cells, can be used.

Inhibitors of human inositol polyphosphate 5-phosphatase also can bedesigned as described in Safrany et al., Biochem. 33, 10763-69, 1994.

Pharmaceutical Compositions

The invention also provides pharmaceutical compositions which can beadministered to a patient to achieve a therapeutic effect.Pharmaceutical compositions of the invention can comprise, for example,a inositol polyphosphate 5-phosphatase polypeptide, inositolpolyphosphate 5-phosphatase polynucleotide, ribozymes or antisenseoligonucleotides, antibodies which specifically bind to a inositolpolyphosphate 5-phosphatase polypeptide, or mimetics, activators, orinhibitors of a inositol polyphosphate 5-phosphatase polypeptideactivity. The compositions can be administered alone or in combinationwith at least one other agent, such as stabilizing compound, which canbe administered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions can be administered to a patient alone, or incombination with other agents, drugs or hormones.

In addition to the active ingredients, these pharmaceutical compositionscan contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically.Pharmaceutical compositions of the invention can be administered by anynumber of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, intraperitoneal,intranasal, parenteral, topical, sublingual, or rectal means.Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents can be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores can be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which also can contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments can be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds canbe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration canbe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions cancontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds can be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Non-lipid polycationicamino polymers also can be used for delivery. Optionally, the suspensionalso can contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions. For topical or nasal administration, penetrantsappropriate to the particular barrier to be permeated are used in theformulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention can bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes. Thepharmaceutical composition can be provided as a salt and can be formedwith many acids, including but not limited to, hydrochloric, sulfuric,acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be moresoluble in aqueous or other protonic solvents than are the correspondingfree base forms. In other cases, the preferred preparation can be alyophilized powder which can contain any or all of the following: 1-50mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5to 5.5, that is combined with buffer prior to use.

Further details on techniques for formulation and administration can befound in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES(Maack Publishing Co., Easton, Pa.). After pharmaceutical compositionshave been prepared, they can be placed in an appropriate container andlabeled for treatment of an indicated condition. Such labeling wouldinclude amount, frequency, and method of administration.

Therapeutic Indications and Methods

Human inositol polyphosphate 5-phosphatase can be regulated to treatCOPD, cancer, diabetes, and asthma.

Cancer is a disease fundamentally caused by oncogenic cellulartransformation. There are several hallmarks of transformed cells thatdistinguish them from their normal counterparts and underlie thepathophysiology of cancer. These include uncontrolled cellularproliferation, unresponsiveness to normal death-inducing signals(immortalization), increased cellular motility and invasiveness,increased ability to recruit blood supply through induction of new bloodvessel formation (angiogenesis), genetic instability, and dysregulatedgene expression. Various combinations of these aberrant physiologies,along with the acquisition of drug-resistance frequently lead to anintractable disease state in which organ failure and patient deathultimately ensue.

Most standard cancer therapies target cellular proliferation and rely onthe differential proliferative capacities between transformed and normalcells for their efficacy. This approach is hindered by the facts thatseveral important normal cell types are also highly proliferative andthat cancer cells frequently become resistant to these agents. Thus, thetherapeutic indices for traditional anti cancer therapies rarely exceed2.0.

The advent of genomics-driven molecular target identification has openedup the possibility of identifying new cancer-specific targets fortherapeutic intervention that will provide safer, more effectivetreatments for cancer patients. Thus, newly discovered tumor-associatedgenes and their products can be tested for their role(s) in disease andused as tools to discover and develop innovative therapies. Genesplaying important roles in any of the physiological processes outlinedabove can be characterized as cancer targets.

Genes or gene fragments identified through genomics can readily beexpressed in one or more heterologous expression systems to producefunctional recombinant proteins. These proteins are characterized invitro for their biochemical properties and then used as tools inhigh-throughput molecular screening programs to identify chemicalmodulators of their biochemical activities. Activators and/or inhibitorsof target protein activity can be identified in this manner andsubsequently tested in cellular and in vivo disease models foranti-cancer activity. Optimization of lead compounds with iterativetesting in biological models and detailed pharmacokinetic andtoxicological analyses form the basis for drug development andsubsequent testing in humans.

Allergy is a complex process in which environmental antigens induceclinically adverse reactions. The inducing antigens, called allergens,typically elicit a specific IgE response and, although in most cases theallergens themselves have little or no intrinsic toxicity, they inducepathology when the IgE response in turn elicits an IgE-dependent or Tcell-dependent hypersensitivity reaction. Hypersensitivity reactions canbe local or systemic and typically occur within minutes of allergenexposure in individuals who have previously been sensitized to anallergen. The hypersensitivity reaction of allergy develops when theallergen is recognized by IgE antibodies bound to specific receptors onthe surface of effector cells, such as mast cells, basophils, oreosinophils, which causes the activation of the effector cells and therelease of mediators that produce the acute signs and symptoms of thereactions. Allergic diseases include asthma, allergic rhinitis (hayfever), atopic dermatitis, and anaphylaxis.

Asthma is though to arise as a result of interactions between multiplegenetic and environmental factors and is characterized by three majorfeatures: 1) intermittent and reversible airway obstruction caused bybronchoconstriction, increased mucus production, and thickening of thewalls of the airways that leads to a narrowing of the airways, 2) airwayhyperresponsiveness caused by a decreased control of airway caliber, and3) airway inflammation. Certain cells are critical to the inflammatoryreaction of asthma and they include T cells and antigen presentingcells, B cells that produce IgE, and mast cells, basophils, eosinophils,and other cells that bind IgE. These effector cells accumulate at thesite of allergic reaction in the airways and release toxic products thatcontribute to the acute pathology and eventually to the tissuedestruction related to the disorder. Other resident cells, such assmooth muscle cells, lung epithelial cells, mucus-producing cells, andnerve cells may also be abnormal in individuals with asthma and maycontribute to the pathology. While the airway obstruction of asthma,presenting clinically as an intermittent wheeze and shortness of breath,is generally the most pressing symptom of the disease requiringimmediate treatment, the inflammation and tissue destruction associatedwith the disease can lead to irreversible changes that eventually makeasthma a chronic disabling disorder requiring long-term management.

Despite recent important advances in our understanding of thepathophysiology of asthma, the disease appears to be increasing inprevalence and severity (Gergen and Weiss, Am. Rev. Respir. Dis. 146,823-24, 1992). It is estimated that 3040% of the population suffer withatopic allergy, and 15% of children and 5% of adults in the populationsuffer from asthma (Gergen and Weiss, 1992). Thus, an enormous burden isplaced on our health care resources. However, both diagnosis andtreatment of asthma are difficult. The severity of lung tissueinflammation is not easy to measure and the symptoms of the disease areoften indistinguishable from those of respiratory infections, chronicrespiratory inflammatory disorders, allergic rhinitis, or otherrespiratory disorders. Often, the inciting allergen cannot bedetermined, making removal of the causative environmental agentdifficult. Current pharmacological treatments suffer their own set ofdisadvantages. Commonly used therapeutic agents, such as betaactivators, can act as symptom relievers to transiently improvepulmonary function, but do not affect the underlying inflammation.Agents that can reduce the underlying inflammation, such asanti-inflammatory steroids, can have major drawbacks that range fromimmunosuppression to bone loss (Goodman and Gilman's THE PHARMACOLOGICBASIS OF THERAPEUTICS, Seventh Edition, MacMillan Publishing Company,NY, USA, 1985). In addition, many of the present therapies, such asinhaled corticosteroids, are short-lasting, inconvenient to use, andmust be used often on a regular basis, in some cases for life, makingfailure of patients to comply with the treatment a major problem andthereby reducing their effectiveness as a treatment.

Because of the problems associated with conventional therapies,alternative treatment strategies have been evaluated. Glycophorin A (Chuand Sharom, Cell. Immunol. 145, 223-39, 1992), cyclosporin (Alexander etal., Lancet 339, 324-28, 1992), and a nonapeptide fragment of IL-2(Zav'yalov et al., Immunol. Lett. 31, 285-88, 1992) all inhibitinterleukin-2 dependent T lymphocyte proliferation; however, they areknown to have many other effects. For example, cyclosporin is used as aimmunosuppressant after organ transplantation. While these agents mayrepresent alternatives to steroids in the treatment of asthmatics, theyinhibit interleukin-2 dependent T lymphocyte proliferation andpotentially critical immune functions associated with homeostasis. Othertreatments that block the release or activity of mediators ofbronchochonstriction, such as cromones or anti-leukotrienes, haverecently been introduced for the treatment of mild asthma, but they areexpensive and not effective in all patients and it is unclear whetherthey have any effect on the chronic changes associated with asthmaticinflammation. What is needed in the art is the identification of atreatment that can act in pathways critical to the development ofasthma_that both blocks the episodic attacks of the disorder andpreferentially dampens the hyperactive allergic immune response withoutimmunocompromising the patient.

Chronic obstructive pulmonary (or airways) disease (COPD) is a conditiondefined physiologically as airflow obstruction that generally resultsfrom a mixture of emphysema and peripheral airway obstruction due tochronic bronchitis (Senior & Shapiro, Pulmonary Diseases and Disorders,3d ed., New York, McGraw-Hill, 1998, pp. 659-681, 1998; Barnes, Chest117, 10S-14S, 2000). Emphysema is characterized by destruction ofalveolar walls leading to abnormal enlargement of the air spaces of thelung. Chronic bronchitis is defined clinically as the presence ofchronic productive cough for three months in each of two successiveyears. In COPD, airflow obstruction is usually progressive and is onlypartially reversible. By far the most important risk factor fordevelopment of COPD is cigarette smoking, although the disease doesoccur in non-smokers.

Chronic inflammation of the airways is a key pathological feature ofCOPD (Senior & Shapiro, 1998). The inflammatory cell populationcomprises increased numbers of macrophages, neutrophils, and CD8⁺lymphocytes. Inhaled irritants, such as cigarette smoke, activatemacrophages which are resident in the respiratory tract, as well asepithelial cells leading to release of chemokines (e.g., interleukin-8)and other chemotactic factors. These chemotactic factors act to increasethe neutrophil/monocyte trafficking from the blood into the lung tissueand airways. Neutrophils and monocytes recruited into the airways canrelease a variety of potentially damaging mediators such as proteolyticenzymes and reactive oxygen species. Matrix degradation and emphysema,along with airway wall thickening, surfactant dysfunction, and mucushypersecretion, all are potential sequelae of this inflammatory responsethat lead to impaired airflow and gas exchange.

Diabetes mellitus is a common metabolic disorder characterized by anabnormal elevation in blood glucose, alterations in lipids andabnormalities (complications) in the cardiovascular system, eye, kidneyand nervous system. Diabetes is divided into two separate diseases: type1 diabetes (juvenile onset), which results from a loss of cells whichmake and secrete insulin, and type 2 diabetes (adult onset), which iscaused by a defect in insulin secretion and a defect in insulin action.

Type 1 diabetes is initiated by an autoimuune reaction that attacks theinsulin secreting cells (beta cells) in the pancreatic islets. Agentsthat prevent this reaction from occurring or that stop the reactionbefore destruction of the beta cells has been accomplished are potentialtherapies for this disease. Other agents that induce beta cellproliferation and regeneration also are potential therapies.

Type II diabetes is the most common of the two diabetic conditions (6%of the population). The defect in insulin secretion is an importantcause of the diabetic condition and results from an inability of thebeta cell to properly detect and respond to rises in blood glucoselevels with insulin release. Therapies that increase the response by thebeta cell to glucose would offer an important new treatment for thisdisease.

The defect in insulin action in Type II diabetic subjects is anothertarget for therapeutic intervention. Agents that increase the activityof the insulin receptor in muscle, liver, and fat will cause a decreasein blood glucose and a normalization of plasma lipids. The receptoractivity can be increased by agents that directly stimulate the receptoror that increase the intracellular signals from the receptor. Othertherapies can directly activate the cellular end process, i.e. glucosetransport or various enzyme systems, to generate an insulin-like effectand therefore a produce beneficial outcome. Because overweight subjectshave a greater susceptibility to Type II diabetes, any agent thatreduces body weight is a possible therapy.

Both Type I and Type diabetes can be treated with agents that mimicinsulin action or that treat diabetic complications by reducing bloodglucose levels. Human inositol polyphosphate 5-phosphatase is expressedin islets (FIG. 16). It therefore represents a potential target for thetreatment of diabetes. Likewise, agents that reduces new blood vesselgrowth can be used to treat the eye complications that develop in bothdiseases.

This invention further pertains to the use of novel agents identified bythe screening assays described above. Accordingly, it is within thescope of this invention to use a test compound identified as describedherein in an appropriate animal model. For example, an agent identifiedas described herein (e.g. a modulating agent, an antisense nucleic acidmolecule, a specific antibody, ribozyme, or a inositol polyphosphate5-phosphatase polypeptide binding molecule) can be used in an animalmodel to determine the efficacy, toxicity, or side effects of treatmentwith such an agent. Alternatively, an agent identified as describedherein can be used in an animal model to determine the mechanism ofaction of such an agent. Furthermore, this invention pertains to uses ofnovel agents identified by the above-described screening assays fortreatments as described herein.

A reagent which affects inositol polyphosphate 5-phosphatase activitycan be administered to a human cell, either in vitro or in vivo, toreduce inositol polyphosphate 5-phosphatase activity. The reagentpreferably binds to an expression product of a human inositolpolyphosphate 5-phosphatase gene. If the expression product is aprotein, the reagent is preferably an antibody. For treatment of humancells ex vivo, an antibody can be added to a preparation of stem cellswhich have been removed from the body. The cells can then be replaced inthe same or another human body, with or without clonal propagation, asis known in the art.

In one embodiment, the reagent is delivered using a liposome.Preferably, the liposome is stable in the animal into which it has beenadministered for at least about 30 minutes, more preferably for at leastabout 1 hour, and even more preferably for at least about 24 hours. Aliposome comprises a lipid composition that is capable of targeting areagent, particularly a polynucleotide, to a particular site in ananimal, such as a human. Preferably, the lipid composition of theliposome is capable of targeting to a specific organ of an animal, suchas the lung, liver, spleen, heart brain, lymph nodes, and skin.

A liposome useful in the present invention comprises a lipid compositionthat is capable of fusing with the plasma membrane of the targeted cellto deliver its contents to the cell. Preferably, the transfectionefficiency of a liposome is about 0.5 μg of DNA per 16 mmole of liposomedelivered to about 10⁶ cells, more preferably about 1.0 μg of DNA per 16mole of liposome delivered to about 10⁶ cells, and even more preferablyabout 2.0 μg of DNA per 16 mmol of liposome delivered to about 10⁶cells. Preferably, a liposome is between about 100 and 500 nm, morepreferably between about 150 and 450 nm, and even more preferablybetween about 200 and 400 nm in diameter.

Suitable liposomes for use in the present invention include thoseliposomes standardly used in, for example, gene delivery methods knownto those of skill in the art. More preferred liposomes include liposomeshaving a polycationic lipid composition and/or liposomes having acholesterol backbone conjugated to polyethylene glycol. Optionally, aliposome comprises a compound capable of targeting the liposome to aparticular cell type, such as a cell-specific ligand exposed on theouter surface of the liposome.

Complexing a liposome with a reagent such as an antisenseoligonucleotide or ribozyme can be achieved using methods which arestandard in the art (see, for example, U.S. Pat. No. 5,705,151).Preferably, from about 0.1 μg to about 10 μg of polynucleotide iscombined with about 8 nmol of liposomes, more preferably from about 0.5μg to about 5 μg of polynucleotides are combined with about 8 mmolliposomes, and even more preferably about 1.0 μg of polynucleotides iscombined with about 8 mmol liposomes.

In another embodiment, antibodies can be delivered to specific tissuesin vivo using receptor-mediated targeted delivery. Receptor-mediated DNAdelivery techniques are taught in, for example, Findeis et al. Trends inBiotechnol. 11, 202-05 (1993); Chiou et al., GENE THERAPEUTICS: METHODSAND APPLICATIONS OF DIRECT GENE TRANSFER (J. A. Wolff, ed.) (1994); Wu &Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al., J. Biol. Chem. 269,54246 (1994); Zenke et al., Proc. Natl. Acad. Sci. USA. 87, 3655-59(1990); Wu et al., J. Biol. Chem 266, 338-42 (1991).

Determination of a Therapeutically Effective Dose

The determination of a therapeutically effective dose is well within thecapability of those skilled in the art. A therapeutically effective doserefers to that amount of active ingredient which increases or decreasesinositol polyphosphate 5-phosphatase activity relative to the inositolpolyphosphate 5-phosphatase activity which occurs in the absence of thetherapeutically effective dose.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models, usuallymice, rabbits, dogs, or pigs. The animal model also can be used todetermine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans.

Therapeutic efficacy and toxicity, e.g., ED₅₀ (the dose therapeuticallyeffective in 50% of the population) and LD₅₀ (the dose lethal to 50% ofthe population), can be determined by standard pharmaceutical proceduresin cell cultures or experimental animals. The dose ratio of toxic totherapeutic effects is the therapeutic index, and it can be expressed asthe ratio, ID₅₀/ED₅₀.

Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activeingredient or to maintain the desired effect. Factors which can be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions can be administered every 3 to 4 days, everyweek, or once every two weeks depending on the half-life and clearancerate of the particular formulation.

Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

If the reagent is a single-chain antibody, polynucleotides encoding theantibody can be constructed and introduced into a cell either ex vivo orin vivo using well-established techniques including, but not limited to,transferrin-polycation-mediated DNA transfer, transfection with naked orencapsulated nucleic acids, liposome-mediated cellular fusion,intracellular transportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, “gene gun,” and DEAE- orcalcium phosphate-mediated transfection.

Effective in vivo dosages of an antibody are in the range of about 5 μgto about 50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μg to about500 μg/kg of patient body weight, and about 200 to about 250 μg/kg ofpatient body weight. For administration of polynucleotides encodingsingle-chain antibodies, effective in vivo dosages are in the range ofabout 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μg to about2 mg, about 51 g to about 500 μg, and about 20 μg to about 100 μg ofDNA.

If the expression product is mRNA, the reagent is preferably anantisense oligonucleotide or a ribozyme. Polynucleotides which expressantisense oligonucleotides or ribozymes can be introduced into cells bya variety of methods, as described above.

Preferably, a reagent reduces expression of a inositol polyphosphate5-phosphatase gene or the activity of a inositol polyphosphate5-phosphatase polypeptide by at least about 10, preferably about 50,more preferably about 75, 90, or 100% relative to the absence of thereagent. The effectiveness of the mechanism chosen to decrease the levelof expression of a inositol polyphosphate 5-phosphatase gene or theactivity of a inositol polyphosphate 5-phosphatase polypeptide can beassessed using methods well known in the art, such as hybridization ofnucleotide probes to inositol polyphosphate 5-phosphatase-specific mRNA,quantitative RT-PCR, immunologic detection of a inositol polyphosphate5-phosphatase polypeptide, or measurement of inositol polyphosphate5-phosphatase activity.

In any of the embodiments described above, any of the pharmaceuticalcompositions of the invention can be administered in combination withother appropriate therapeutic agents. Selection of the appropriateagents for use in combination therapy can be made by one of ordinaryskill in the art, according to conventional pharmaceutical principles.The combination of therapeutic agents can act synergistically to effectthe treatment or prevention of the various disorders described above.Using this approach, one may be able to achieve therapeutic efficacywith lower dosages of each agent, thus reducing the potential foradverse side effects.

Any of the therapeutic methods described above can be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

Diagnostic Methods

Human inositol polyphosphate 5-phosphatase also can be used indiagnostic assays for detecting diseases and abnormalities orsusceptibility to diseases and abnormalities related to the presence ofmutations in the nucleic acid sequences which encode the enzyme. Forexample, differences can be determined between the cDNA or genomicsequence encoding inositol polyphosphate 5-phosphatase in individualsafflicted with a disease and in normal individuals. If a mutation isobserved in some or all of the afflicted individuals but not in normalindividuals, then the mutation is likely to be the causative agent ofthe disease.

Sequence differences between a reference gene and a gene havingmutations can be revealed by the direct DNA sequencing method. Inaddition, cloned DNA segments can be employed as probes to detectspecific DNA segments. The sensitivity of this method is greatlyenhanced when combined with PCR. For example, a sequencing primer can beused with a double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures using radiolabeled nucleotides orby automatic sequencing procedures using fluorescent tags.

Genetic testing based on DNA sequence differences can be carried out bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized, for example, by high resolution gelelectrophoresis. DNA fragments of different sequences can bedistinguished on denaturing formamide gradient gels in which themobilities of different DNA fragments are retarded in the gel atdifferent positions according to their specific melting or partialmelting temperatures (see, e.g., Myers et al., Science 230, 1242, 1985).Sequence changes at specific locations can also be revealed by nucleaseprotection assays, such as RNase and S 1 protection or the chemicalcleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci. USA 85,4397-4401, 1985). Thus, the detection of a specific DNA sequence can beperformed by methods such as hybridization, RNase protection, chemicalcleavage, direct DNA sequencing or the use of restriction enzymes andSouthern blotting of genomic DNA. In addition to direct methods such asgel-electrophoresis and DNA sequencing, mutations can also be detectedby in situ analysis.

Altered levels of a inositol polyphosphate 5-phosphatase also can bedetected in various tissues. Assays used to detect levels of thereceptor polypeptides in a body sample, such as blood or a tissuebiopsy, derived from a host are well known to those of skill in the artand include radioimmunoassays, competitive binding assays, Western blotanalysis, and ELISA assays.

All patents and patent applications cited in this disclosure areexpressly incorporated herein by reference. The above disclosuregenerally describes the present invention. A more complete understandingcan be obtained by reference to the following specific examples whichare provided for purposes of illustration only and are not intended tolimit the scope of the invention.

EXAMPLE 1

Detection of Inositol Polyphosphate 5-phosphatase Activity

The polynucleotide of SEQ ID NO: 1 is inserted into the expressionvector pCEV4 and the expression vector pCEV4-inositol polyphosphate5-phosphatase polypeptide obtained is transfected into human embryonickidney 293 cells. From these cells extracts are obtained and inositolpolyphosphate 5-phosphatase activity is carried out using [3H]Ins(1, 3,4)P3, [3H]Ins(1, 4, 5)P3, and [3H]Ins(1, 3, 4, 5)P4. The separation ofinositol polyphosphates by high performance liquid chromatography isdone according to the method of Zhang and Buxton. The flow rate is 0.6ml/min, and each fraction is collected for 30 s. Collected samples arediluted to 1/20 volume with distilled water and quantitated by liquidscintillation counting. For the assay of Ins(1,3,4)P3 hydrolyzingactivity, the D-5 position phosphate of Ins (1, 3, 4, 5)P4 is hydrolyzedby recombinant SHIP 1 protein and used as a substrate. The SHIP 1protein is prepared the same way as the present inositol polyphosphate5-phosphatase polypeptide. The PtdIns(4,5)P2 phosphatase activity isdetermined. The spots on the thin layer chromatography plate arevisualized by exposing the plate to x-ray film (Eastman Kodak ScientificCo., Rochester, N.Y.) for 6 days at −80° C. Before the exposure, theintensity of the radioactivity was enhanced by EN3HANCE spray (NEN LifeScience Products). It is shown that the polypeptide of SEQ ID NO: 2 hasa inositol polyphosphate 5-phosphatase activity.

EXAMPLE 2

Expression of Recombinant Human Inositol Polyphosphate 5-phosphatase

The Pichia pastoris expression vector pPICZB (Invitrogen, San Diego,Calif.) is used to produce large quantities of recombinant humaninositol polyphosphate 5-phosphatase polypeptides in yeast. The inositolpolyphosphate 5-phosphatase-encoding DNA sequence is derived from SEQ IDNOS:1 AND 11. Before insertion into vector pPICZB, the DNA sequence ismodified by well known methods in such a way that it contains at its5′-end an initiation codon and at its 3′-end an enterokinase cleavagesite, a His6 reporter tag and a termination codon. Moreover, at bothtermini recognition sequences for restriction endonucleases are addedand after digestion of the multiple cloning site of pPICZ B with thecorresponding restriction enzymes the modified DNA sequence is ligatedinto pPICZB. This expression vector is designed for inducible expressionin Pichia pastoris, driven by a yeast promoter. The resultingpPICZ/md-His6 vector is used to transform the yeast.

The yeast is cultivated under usual conditions in 5 liter shake flasksand the recombinantly produced protein isolated from the culture byaffinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea. Thebound polypeptide is eluted with buffer, pH 3.5, and neutralized.Separation of the polypeptide from the His6 reporter tag is accomplishedby site-specific proteolysis using enterokinase (Invitrogen, San Diego,Calif.) according to manufacturer's instructions. Purified humaninositol polyphosphate 5-phosphatase polypeptide is obtained.

EXAMPLE 3

Identification of Test Compounds that Bind to Inositol polyphosphate5-phosphatase Polypeptides

Purified inositol polyphosphate 5-phosphatase polypeptides comprising aglutathione-S-transferase protein and absorbed ontoglutathione-derivatized wells of 96-well microtiter plates are contactedwith test compounds from a small molecule library at pH 7.0 in aphysiological buffer solution. Human inositol polyphosphate5-phosphatase polypeptides comprise the amino acid sequence shown in SEQID NOS:2 AND 12. The test compounds comprise a fluorescent tag. Thesamples are incubated for 5 minutes to one hour. Control samples areincubated in the absence of a test compound.

The buffer solution containing the test compounds is washed from thewells. Binding of a test compound to a inositol polyphosphate5-phosphatase polypeptide is detected by fluorescence measurements ofthe contents of the wells. A test compound which increases thefluorescence in a well by at least 15% relative to fluorescence of awell in which a test compound is not incubated is identified as acompound which binds to a inositol potyphosphate 5-phosphatasepolypeptide.

EXAMPLE 4

Identification of a Test Compound which Decreases Inositol Polyphosphate5-phosphatase Gene Expression

A test compound is administered to a culture of human cells transfectedwith a inositol polyphosphate 5-phosphatase expression construct andincubated at 37° C. for 10 to 45 minutes. A culture of the same type ofcells which have not been transfected is incubated for the same timewithout the test compound to provide a negative control.

RNA is isolated from the two cultures as described in Chirgwin et al.,Biochem. 18, 5294-99, 1979). Northern blots are prepared using 20 to 30μg total RNA and hybridized with a ³²P-labeled inositol polyphosphate5-phosphatase-specific probe at 65° C. in Express-hyb (CLONTECH). Theprobe comprises at least 11 contiguous nucleotides selected from thecomplement of SEQ ID NOS:1 AND 11. A test compound which decreases theinositol polyphosphate 5-phosphatase-specific signal relative to thesignal obtained in the absence of the test compound is identified as aninhibitor of inositol polyphosphate 5-phosphatase gene expression.

EXAMPLE 5

Identification of a Test Compound which Decreases Inositol Polyphosphate5-phosphatase Activity

A test compound is administered to a culture of human cells transfectedwith a inositol polyphosphate 5-phosphatase expression construct andincubated at 370° C. for 10 to 45 minutes. A culture of the same type ofcells which have not been transfected is incubated for the same timewithout the test compound to provide a negative control. Enzyme activityis measured using the method described in U.S. Pat. No. 6,090,621.Inositol polyphosphate 5-phosphatases hydrolyze the 5 phosphate fromIns(1,4,5)P3 and Ins(1,3,4,5)P₄; a subset of these enzymes can removethe 5-phosphate from phosphatidylinositol polyphosphates. To determinewhether the enzyme of the invention is a functional inositol phosphatasein the presence of various test compounds, anti-human inositolpolyphosphate 5-phosphatase immunoprecipitates are assayed for theirability to hydrolyze inositol phosphates and phosphatidylinositolpolyphosphates.

A test compound which decreases the enzyme activity of the inositolpolyphosphate 5-phosphatase relative to the enzyme activity in theabsence of the test compound is identified as an inhibitor of inositolpolyphosphate 5-phosphatase activity.

EXAMPLE 6

Tissue-Specific Expression of Human Inositol Polyphosphate 5-phosphatase

The qualitative expression pattern of inositol polyphosphate5-phosphatase in various tissues is determined by ReverseTranscription-Polymerase Chain Reaction (RT-PCR). To demonstrate thatinositol polyphosphate 5-phosphatase is involved in the disease processof asthma, the following whole body panel is screened to showpredominant or relatively high expression in lung or immune tissues:brain, heart, kidney, liver, lung, trachea, bone marrow, colon, smallintestine, spleen, stomach, thymus, mammary gland, skeletal muscle,prostate, testis, uterus, cerebellum, fetal brain, fetal liver, spinalcord, placenta, adrenal gland, pancreas, salivary gland, thyroid,peripheral blood leukocytes, lymph node, and tonsil. Once this isestablished, the following lung and immune system cells are screened tolocalize expression to particular cell subsets: lung microvascularendothelial cells, bronchial/tracheal epithelial cells,bronchial/tracheal smooth muscle cells, lung fibroblasts, T cells (Thelper 1 subset, T helper 2 subset, NKT cell subset, and cytotoxic Tlymphocytes), B cells, mononuclear cells (monocytes and macrophages),mast cells, eosinophils, neutrophils, and dendritic cells. As a finalstep, the expression of inositol polyphosphate 5-phosphatase in cellsderived from normal individuals with the expression of cells derivedfrom asthmatic individuals is compared.

To demonstrate that inositol polyphosphate 5-phosphatase is involved incancer, expression is determined in the following tissues: adrenalgland, bone marrow, brain, cerebellum, colon, fetal brain, fetal liver,heart, kidney, liver, lung, mammary gland, pancreas, placenta, prostate,salivary gland, skeletal muscle, small intestine, spinal cord, spleen,stomach, testis, thymus, thyroid, trachea, uterus, and peripheral bloodlymphocytes. Expression in the following cancer cell lines also isdetermined: DU-145 (prostate), NCI-H125 (lung), HT-29 (colon), COLO-205(colon), A-549 (lung), NCI-H460 (lung), HT-116 (colon), DLD-1 (colon),MDA-MD-231 (breast), LS174T (colon), ZF-75 (breast), MDA-MN-435(breast), HT-1080, MCF-7 (breast), and U87. Matched pairs of malignantand normal tissue from the same patient also are tested.

To demonstrate that inositol polyphosphate 5-phosphatase is involved inthe disease process of COPD, the initial expression panel consists ofRNA samples from respiratory tissues and inflammatory cells relevant toCOPD: lung (adult and fetal), trachea, freshly isolated alveolar type IIcells, cultured human bronchial epithelial cells, cultured small airwayepithelial cells, cultured bronchial sooth muscle cells, cultured H441cells (Clara-like), freshly isolated neutrophils and monocytes, andcultured monocytes (macrophage-like). Body map profiling also is carriedout, using total RNA panels purchased from Clontech. The tissues areadrenal gland, bone marrow, brain, colon, heart, kidney, liver, lung,mammary gland, pancreas, prostate, salivary gland, skeletal muscle,small intestine, spleen, stomach, testis, thymus, trachea, thyroid, anduterus. As a final step, the expression of human inositol polyphosphate5-phosphatase in cells derived from normal individuals with theexpression of cells derived from COPD-affected individuals is compared.

To demonstrate that human inositol polyphosphate 5-phosphatase isinvolved in the disease process of diabetes, the following whole bodypanel is screened to show predominant or relatively high expression:subcutaneous and mesenteric adipose tissue, adrenal gland, bone marrow,brain, colon, fetal brain, heart, hypothalamus, kidney, liver, lung,mammary gland, pancreas, placenta, prostate, salivary gland, skeletalmuscle, small intestine, spleen, stomach, testis, thymus, thyroid,trachea, and uterus. Human islet cells and an islet cell library alsoare tested. As a final step, the expression of human inositolpolyphosphate 5-phosphatase in cells derived from normal individualswith the expression of cells derived from diabetic individuals iscompared.

Quantitative expression profiling. Quantitative expression profiling isperformed by the form of quantitative PCR analysis called “kineticanalysis” firstly described in Higuchi et al., BioTechnology 10, 413-17,1992, and Higuchi et al., BioTechnology 11, 1026-30, 1993. The principleis that at any given cycle within the exponential phase of PCR, theamount of product is proportional to the initial number of templatecopies.

If the amplification is performed in the presence of an internallyquenched fluorescent oligonucleotide (TaqMan probe) complementary to thetarget sequence, the probe is cleaved by the 5′-3′ endonuclease activityof Taq DNA polymerase and a fluorescent dye released in the medium(Holland et al., Proc. Natl. Acad. Sci. U.S.A. 88, 7276-80, 1991).Because the fluorescence emission will increase in direct proportion tothe amount of the specific amplified product, the exponential growthphase of PCR product can be detected and used to determine the initialtemplate concentration (Heid et al., Genome Res. 6, 986-94, 1996, andGibson et al., Genome Res. 6,995-1001, 1996).

The amplification of an endogenous control can be performed tostandardize the amount of sample RNA added to a reaction. In this kindof experiment, the control of choice is the 18S ribosomal RNA. Becausereporter dyes with differing emission spectra are available, the targetand the endogenous control can be independently quantified in the sametube if probes labeled with different dyes are used.

All “real time PCR” measurements of fluorescence are made in the ABIPrism 7700.

RNA extraction and cDNA preparation. Total RNA from the tissues listedabove are used for expression quantification. RNAs labeled “fromautopsy” were extracted from autoptic tissues with the TRIzol reagent(Life Technologies, MD) according to the manufacturer's protocol.

Fifty μg of each RNA were treated with DNase I for 1 hour at 37° C. inthe following reaction mix: 0.2 U/μl RNase-free DNase I (RocheDiagnostics, Germany); 0.4 U/μl RNase inhibitor (PE Applied Biosystems,CA); 10 mM Tris-HCl pH 7.9; 10 mM MgCl₂; 50 mM NaCl; and 1 mM DTT.

After incubation, RNA is extracted once with 1 volume ofphenol:chloroform:—isoamyl alcohol (24:24:1) and once with chloroform,and precipitated with 1/10 volume of 3 M NaAcetate, pH 5.2, and 2volumes of ethanol.

Fifty μg of each RNA from the autoptic tissues are DNase treated withthe DNA-free kit purchased from Ambion (Ambion, TX). After resuspensionand spectro-photometric quantification, each sample is reversetranscribed with the TaqMan Reverse Transcription Reagents (PE AppliedBiosystems, CA) according to the manufacturer's protocol. The finalconcentration of RNA in the reaction mix is 200 ng/μL. Reversetranscription is carried out with 2.5 of random hexamer primers.

TaqMan quantitative analysis. Specific primers and probe are designedaccording to the recommendations of PE Applied Biosystems. Probes can belabeled at the 5′ end with FAM (6-carboxyfluorescein) and at the 3′ endwith TAMRA (6-carboxyt-tramethylrhodamine. Quantification experimentsare performed on 10 ng of reverse transcribed RNA from each sample. Eachdetermination is done in triplicate.

Total cDNA content is normalized with the simultaneous quantification(multiplex PCR) of the 18S ribosomal RNA using the Pre-Developed TaqManAssay Reagents (PDAR) Control Kit (PE Applied Biosystems, CA).

The assay reaction mix is as follows: IX final TaqMan Universal PCRMaster Mix (from 2× stock) (PE Applied Biosystems, CA); 1×PDARcontrol—18S RNA (from 20× stock); 300 nM forward primer; 900 nM reverseprimer; 200 nM probe; 10 ng cDNA; and water to 25 ml.

Each of the following steps are carried out once: pre PCR, 2 minutes at50° C., and 10 minutes at 95° C. The following steps are carried out 40times: denaturation, 15 seconds at 95° C., annealing extension, 1 minuteat 60° C.

The experiment is performed on an ABI Prism 7700 Sequence Detector (PEApplied Biosystems, CA). At the end of the run, fluorescence dataacquired during PCR are processed as described in the ABI Prism 7700user's manual in order to achieve better background subtraction as wellas signal linearity with the starting target quantity.

EXAMPLE 7

Diabetes: In Vivo Testing of Compounds/Target Validation

1. Glucose Production

Over-production of glucose by the liver, due to an enhanced rate ofgluconeogenesis, is the major cause of fasting hyperglycemia indiabetes. Overnight fasted normal rats or mice have elevated rates ofgluconeogenesis as do streptozotocin-induced diabetic rats or mice fedad libitum. Rats are made diabetic with a single intravenous injectionof 40 mg/kg of streptozotocin while C57BL/KsJ mice are given 40-60 mg/kgi.p. for 5 consecutive days. Blood glucose is measured from tail-tipblood and then compounds are administered via different routes p.o.,i.p., i.v., s.c.). Blood is collected at various times thereafter andglucose measured. Alternatively, compounds are administered for severaldays, then the animals are fasted overnight, blood is collected andplasma glucose measured. Compounds that inhibit glucose production willdecrease plasma glucose levels compared to the vehicle-treated controlgroup.

2. Insulin Sensitivity

Both ob/ob and db/db mice as well as diabetic Zucker rats arehyperglycemic, hyperinsulinemic and insulin resistant. The animals arepre-bled, their glucose levels measured, and then they are grouped sothat the mean glucose level is the same for each group. Compounds areadministered daily either q.d. or b.i.d. by different routes (p.o.,i.p., s.c.) for 7-28 days. Blood is collected at various times andplasma glucose and insulin levels determined. Compounds that improveinsulin sensitivity in these models will decrease both plasma glucoseand insulin levels when compared to the vehicle-treated control group.

3. Insulin Secretion

Compounds that enhance insulin secretion from the pancreas will increaseplasma insulin levels and improve the disappearance of plasma glucosefollowing the administration of a glucose load. When measuring insulinlevels, compounds are administered by different routes (p.o., i.p., s.c.or i.v.) to overnight fasted normal rats or mice. At the appropriatetime an intravenous glucose load (0.4 g/kg) is given, blood is collectedone minute later. Plasma insulin levels are determined. Compounds thatenhance insulin secretion will increase plasma insulin levels comparedto animals given only glucose. When measuring glucose disappearance,animals are bled at the appropriate time after compound administration,then given either an oral or intraperitoneal glucose load (1 g/kg), bledagain after 15, 30, 60 and 90 minutes and plasma glucose levelsdetermined. Compounds that increase insulin levels will decrease glucoselevels and the area-under-the glucose curve when compared to thevehicle-treated group given only glucose.

Compounds that enhance insulin secretion from the pancreas will increaseplasma insulin levels and improve the disappearance of plasma glucosefollowing the administration of a glucose load. When measuring insulinlevels, test compounds which regulate pristanoyl-CoA oxidase-like enzymeare administered by different routes (p.o., i.p., s.c., or i.v.) toovernight fasted normal rats or mice. At the appropriate time anintravenous glucose load (0.4 g/kg) is given, blood is collected oneminute later. Plasma insulin levels are determined. Test compounds thatenhance insulin secretion will increase plasma insulin levels comparedto animals given only glucose. When measuring glucose disappearance,animals are bled at the appropriate time after compound administration,then given either an oral or intraperitoneal glucose load (1 g/kg), bledagain after 15, 30, 60, and 90 minutes and plasma glucose levelsdetermined. Test compounds that increase insulin levels will decreaseglucose levels and the area-under-the glucose curve when compared to thevehicle-treated group given only glucose.

4. Glucose Production

Over-production of glucose by the liver, due to an enhanced rate ofgluconeogenesis, is the major cause of fasting hyperglycemia indiabetes. Overnight fasted normal rats or mice have elevated rates ofgluconeogenesis as do streptozotocin-induced diabetic rats or mice fedad libitum. Rats are made diabetic with a single intravenous injectionof 40 mg/kg of streptozotocin while C57BL/KsJ mice are given 40-60 mg/kgi.p. for 5 consecutive days. Blood glucose is measured from tail-tipblood and then compounds are administered via different routes (p.o.,i.p., i.v., s.c.). Blood is collected at various times thereafter andglucose measured. Alternatively, compounds are administered for severaldays, then the animals are fasted overnight, blood is collected andplasma glucose measured. Compounds that inhibit glucose production willdecrease plasma glucose levels compared to the vehicle-treated controlgroup.

5. Insulin Sensitivity

Both ob/ob and db/db mice as well as diabetic Zucker rats arehyperglycemic, hyperinsulinemic and insulin resistant. The animals arepre-bled, their glucose levels measured, and then they are grouped sothat the mean glucose level is the same for each group. Compounds areadministered daily either q.d. or b.i.d. by different routes (p.o.,i.p., s.c.) for 7-28 days. Blood is collected at various times andplasma glucose and insulin levels determined. Compounds that improveinsulin sensitivity in these models will decrease both plasma glucoseand insulin levels when compared to the vehicle-treated control group.

6. Insulin Secretion

Compounds that enhance insulin secretion from the pancreas will increaseplasma insulin levels and improve the disappearance of plasma glucosefollowing the administration of a glucose load. When measuring insulinlevels, compounds are administered by different routes (p.o., i.p., s.c.or i.v.) to overnight fasted normal rats or mice. At the appropriatetime an intravenous glucose load (0.4 g/kg) is given, blood is collectedone minute later. Plasma insulin levels are determined. Compounds thatenhance insulin secretion will increase plasma insulin levels comparedto animals given only glucose. When measuring glucose disappearance,animals are bled at the appropriate time after compound administration,then given either an oral or intraperitoneal glucose load (1 g/kg), bledagain after 15, 30, 60 and 90 minutes and plasma glucose levelsdetermined. Compounds that increase insulin levels will decrease glucoselevels and the area-under-the glucose curve when compared to thevehicle-treated group given only glucose.

EXAMPLE 8

Proliferation Inhibition Assay: Antisense Oligonucleotides Suppress theGrowth of Cancer Cell Lines

The cell line used for testing is the human colon cancer cell lineHCT116. Cells are cultured in RPMI-1640 with 10-15% fetal calf serum ata concentration of 10,000 cells per milliliter in a volume of 0.5 ml andkept at 37° C. in a 95% air/5% CO₂ atmosphere.

Phosphorothioate oligoribonucleotides are synthesized on an AppliedBiosystems Model 380B DNA synthesizer using phosphoroamidite chemistry.A sequence of 24 bases complementary to the nucleotides at position 1 to24 of SEQ ID NOS:1 AND 11 is used as the test oligonucleotide. As acontrol, another (random) sequence is used: 5′-TCA ACT GAC TAG ATG TACATG GAC-3′. Following assembly and deprotection, oligonucleotides areethanol-precipitated twice, dried, and suspended in phosphate bufferedsaline at the desired concentration. Purity of the oligonucleotides istested by capillary gel electrophoresis and ion exchange HPLC. Thepurified oligonucleotides are added to the culture medium at aconcentration of 10 μM once per day for seven days.

The addition of the test oligonucleotide for seven days results insignificantly reduced expression of human inositol polyphosphate5-phosphatase as determined by Western blotting. This effect is notobserved with the control oligonucleotide. After 3 to 7 days, the numberof cells in the cultures is counted using an automatic cell counter. Thenumber of cells in cultures treated with the test oligonucleotide(expressed as 100%) is compared with the number of cells in culturestreated with the control oligonucleotide. The number of cells incultures treated with the test oligonucleotide is not more than 30% ofcontrol, indicating that the inhibition of human inositol polyphosphate5-phosphatase has an anti-proliferative effect on cancer cells.

EXAMPLE 9

In Vivo Testing of Compounds/Target Validation

1. Acute Mechanistic Assays

1.1. Reduction in Mitogenic Plasma Hormone Levels

This non-tumor assay measures the ability of a compound to reduce eitherthe endogenous level of a circulating hormone or the level of hormoneproduced in response to a biologic stimulus. Rodents are administeredtest compound (p.o., i.p., i.v., i.m., or s.c.). At a predetermined timeafter administration of test compound, blood plasma is collected. Plasmais assayed for levels of the hormone of interest. If the normalcirculating levels of the hormone are too low and/or variable to provideconsistent results, the level of the hormone may be elevated by apre-treatment with a biologic stimulus (i.e., LHRH may be injected i.m.into mice at a dosage of 30 ng/mouse to induce a burst of testosteronesynthesis). The timing of plasma collection would be adjusted tocoincide with the peak of the induced hormone response. Compound effectsare compared to a vehicle-treated control group. An F-test is preformedto determine if the variance is equal or unequal followed by a Student'st-test. Significance is p value≦0.05 compared to the vehicle controlgroup.

1.2. Hollow Fiber Mechanism of Action Assay

Hollow fibers are prepared with desired cell line(s) and implantedintraperitoneally and/or subcutaneously in rodents. Compounds areadministered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested inaccordance with specific readout assay protocol, these may includeassays for gene expression (bDNA, PCR, or Taqman), or a specificbiochemical activity (i.e., cAMP levels. Results are analyzed byStudent's t-test or Rank Sum test after the variance between groups iscompared by an F-test, with significance at p≦0.05 as compared to thevehicle control group.

2. Subacute Functional In Vivo Assays

2.1 Reduction in Mass of Hormone Dependent Tissues

This is another non-tumor assay that measures the ability of a compoundto reduce the mass of a hormone dependent tissue (ie., seminal vesiclesin males and uteri in females). Rodents are administered test compound(p.o., i.p., i.v., i.m., or s.c.) according to a predetermined scheduleand for a predetermined duration (i.e., 1 week). At termination of thestudy, animals are weighed, the target organ is excised, any fluid isexpressed, and the weight of the organ is recorded. Blood plasma mayalso be collected. Plasma may be assayed for levels of a hormone ofinterest or for levels of test agent. Organ weights may be directlycompared or they may be normalized for the body weight of the animal.Compound effects are compared to a vehicle-treated control group. AnF-test is preformed to determine if the variance is equal or unequalfollowed by a Student's t-test. Significance is p value≦0.05 compared tothe vehicle control group.

2.2. Hollow Fiber Proliferation Assay

Hollow fibers are prepared with desired cell line(s) and implantedintraperitoneally and/or subcutaneously in rodents. Compounds areadministered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested inaccordance with specific readout assay protocol. Cell proliferation isdetermined by measuring a marker of cell number (i.e., MIT or LDH). Thecell number and change in cell number from the starting inoculum areanalyzed by Student's t-test or Rank Sum test after the variance betweengroups is compared by an F-test, with significance at p≦0.05 as comparedto the vehicle control group.

2.3. Anti-Angiogenesis Models

2.3.1. Corneal Angiogenesis

Hydron pellets with or without growth factors or cells are implantedinto a micropocket surgically created in the rodent cornea. Compoundadministration may be systemic or local (compound mixed with growthfactors in the hydron pellet). Corneas are harvested at 7 days postimplantation immediately following intracardiac infusion of colloidalcarbon and are fixed in 10% formalin. Readout is qualitative scoringand/or image analysis. Qualitative scores are compared by Rank Sum test.Image analysis data is evaluated by measuring the area ofneovascularization (in pixels) and group averages are compared byStudent's t-test (2 tail). Significance is p≦0.05 as compared to thegrowth factor or cells only group.

2.3.2. Matrigel Angiogenesis

Matrigel, containing cells or growth factors, is injectedsubcutaneously. Compounds are administered p.o., i.p., i.v., i.m., ors.c. Matrigel plugs are harvested at predetermined time point(s) andprepared for readout. Readout is an ELISA-based assay for hemoglobinconcentration and/or histological examination (i.e. vessel count,special staining for endothelial surface markers: CD31, factor-8).Readouts are analyzed by Student's t-test, after the variance betweengroups is compared by an F-test, with significance determined at p≦0.05as compared to the vehicle control group.

3. Primary Antitumor Efficacy

3.1. Early Therapy Models

3.1.1. Subcutaneous Tumor

Tumor cells or fragments are implanted subcutaneously on Day 0. Vehicleand/or compounds are administered p.o., i.p., i.v., i.m., or s.c.according to a predetermined schedule starting at a time, usually on Day1, prior to the ability to measure the tumor burden. Body weights andtumor measurements are recorded 2-3 times weekly. Mean net body andtumor weights are calculated for each data collection day. Anti-tumorefficacy may be initially determined by comparing the size of treated(T) and control (C) tumors on a given day by a Student's t-test, afterthe variance between groups is compared by an F-test, with significancedetermined at p≦0.05. The experiment may also be continued past the endof dosing in which case tumor measurements would continue to be recordedto monitor tumor growth delay. Tumor growth delays are expressed as thedifference in the median time for the treated and control groups toattain a predetermined size divided by the median time for the controlgroup to attain that size. Growth delays are compared by generatingKaplan-Meier curves from the times for individual tumors to attain theevaluation size. Significance is p≦0.05.

3.1.2. Intraperitoneal/Intracranial Tumor Models

Tumor cells are injected intraperitoneally or intracranially on Day 0.Compounds are administered p.o., i.p., i.v., i.m., or s.c. according toa predetermined schedule starting on Day 1. Observations of morbidityand/or mortality are recorded twice daily. Body weights are measured andrecorded twice weekly. Morbidity/mortality data is expressed in terms ofthe median time of survival and the number of long-term survivors isindicated separately. Survival times are used to generate Kaplan-Meiercurves. Significance is p≦0.05 by a log-rank test compared to thecontrol group in the experiment.

3.2. Established Disease Model

Tumor cells or fragments are implanted subcutaneously and grown to thedesired size for treatment to begin. Once at the predetermined sizerange, mice are randomized into treatment groups. Compounds areadministered p.o., i.p., i.v., i.m., or s.c. according to apredetermined schedule. Tumor and body weights are measured and recorded2-3 times weekly. Mean tumor weights of all groups over days postinoculation are graphed for comparison. An F-test is preformed todetermine if the variance is equal or unequal followed by a Student'st-test to compare tumor sizes in the treated and control groups at theend of treatment. Significance is p≦0.05 as compared to the controlgroup. Tumor measurements may be recorded after dosing has stopped tomonitor tumor growth delay. Tumor growth delays are expressed as thedifference in the median time for the treated and control groups toattain a predetermined size divided by the median time for the controlgroup to attain that size. Growth delays are compared by generatingKaplan-Meier curves from the times for individual tumors to attain theevaluation size. Significance is p value≦0.05 compared to the vehiclecontrol group.

3.3. Orthotopic Disease Models

3.3.1. Mammary Fat Pad Assay

Tumor cells or fragments, of mammary adenocarcinoma origin, areimplanted directly into a surgically exposed and reflected mammary fatpad in rodents. The fat pad is placed back in its original position andthe surgical site is closed. Hormones may also be administered to therodents to support the growth of the tumors. Compounds are administeredp.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule.Tumor and body weights are measured and recorded 2-3 times weekly. Meantumor weights of all groups over days post inoculation are graphed forcomparison. An F-test is preformed to determine if the variance is equalor unequal followed by a Student's t-test to compare tumor sizes in thetreated and control groups at the end of treatment Significance isp≦0.05 as compared to the control group.

Tumor measurements may be recorded after dosing has stopped to monitortumor growth delay. Tumor growth delays are expressed as the differencein the median time for the treated and control groups to attain apredetermined size divided by the median time for the control group toattain that size. Growth delays are compared by generating Kaplan-Meiercurves from the times for individual tumors to attain the evaluationsize. Significance is p value≦0.05 compared to the vehicle controlgroup. In addition, this model provides an opportunity to increase therate of spontaneous metastasis of this type of tumor. Metastasis can beassessed at termination of the study by counting the number of visiblefoci per target organ, or measuring the target organ weight. The meansof these endpoints are compared by Student's t-test after conducting anF-test, with significance determined at p≦0.05 compared to the controlgroup in the experiment.

3.3.2. Intraprostatic Assay

Tumor cells or fragments, of prostatic adenocarcinoma origin, areimplanted directly into a surgically exposed dorsal lobe of the prostatein rodents. The prostate is externalized through an abdominal incisionso that the tumor can be implanted specifically in the dorsal lobe whileverifying that the implant does not enter the seminal vesicles. Thesuccessfully inoculated prostate is replaced in the abdomen and theincisions through the abdomen and skin are closed. Hormones may also beadministered to the rodents to support the growth of the tumors.Compounds are administered p.o., i.p., i.v., i.m., or s.c. according toa predetermined schedule. Body weights are measured and recorded 2-3times weekly. At a predetermined time, the experiment is terminated andthe animal is dissected. The size of the primary tumor is measured inthree dimensions using either a caliper or an ocular micrometer attachedto a dissecting scope. An F-test is preformed to determine if thevariance is equal or unequal followed by a Student's t-test to comparetumor sizes in the treated and control groups at the end of treatment.Significance is p≦0.05 as compared to the control group. This modelprovides an opportunity to increase the rate of spontaneous metastasisof this type of tumor. Metastasis can be assessed at termination of thestudy by counting the number of visible foci per target organ (i.e., thelungs), or measuring the target organ weight (i.e., the regional lymphnodes). The means of these endpoints are compared by Student's t-testafter conducting an F-test, with significance determined at p≦0.05compared to the control group in the experiment.

3.3.3. Intrabronchial Assay

Tumor cells of pulmonary origin may be implanted intrabronchially bymaking an incision through the skin and exposing the trachea The tracheais pierced with the beveled end of a 25 gauge needle and the tumor cellsare inoculated into the main bronchus using a flat-ended 27 gauge needlewith a 90° bend. Compounds are administered p.o., i.p., i.v., i.m., ors.c. according to a predetermined schedule. Body weights are measuredand recorded 2-3 times weekly. At a predetermined time, the experimentis terminated and the animal is dissected. The size of the primary tumoris measured in three dimensions using either a caliper or an ocularmicrometer attached to a dissecting scope. An F-test is preformed todetermine if the variance is equal or unequal followed by a Student'st-test to compare tumor sizes in the treated and control groups at theend of treatment. Significance is p≦0.05 as compared to the controlgroup. This model provides an opportunity to increase the rate ofspontaneous metastasis of this type of tumor. Metastasis can be assessedat termination of the study by counting the number of visible foci pertarget organ (i.e., the contralateral lung), or measuring the targetorgan weight. The means of these endpoints are compared by Student'st-test after conducting an F-test, with significance determined atp≦0.05 compared to the control group in the experiment.

3.3.4. Intracecal Assay

Tumor cells of gastrointestinal origin may be implanted intracecally bymaking an abdominal incision through the skin and externalizing theintestine. Tumor cells are inoculated into the cecal wall withoutpenetrating the lumen of the intestine using a 27 or 30 gauge needle.Compounds are administered p.o., i.p., i.v., i.m., or s.c. according toa predetermined schedule. Body weights are measured and recorded 2-3times weekly. At a predetermined time, the experiment is terminated andthe animal is dissected. The size of the primary tumor is measured inthree dimensions using either a caliper or an ocular micrometer attachedto a dissecting scope. An F-test is preformed to determine if thevariance is equal or unequal followed by a Student's t-test to comparetumor sizes in the treated and control groups at the end of treatment.Significance is p≦0.05 as compared to the control group. This modelprovides an opportunity to increase the rate of spontaneous metastasisof this type of tumor. Metastasis can be assessed at termination of thestudy by counting the number of visible foci per target organ (i.e., theliver), or measuring the target organ weight. The means of theseendpoints are compared by Student's t-test after conducting an F-test,with significance determined at p≦0.05 compared to the control group inthe experiment.

4. Secondary (Metastatic) Antitumor Efficacy

4.1 Spontaneous Metastasis

Tumor cells are inoculated s.c. and the tumors allowed to grow to apredetermined range for spontaneous metastasis studies to the lung orliver. These primary tumors are then excised. Compounds are administeredp.o., i.p., i.v., i.m., or s.c. according to a predetermined schedulewhich may include the period leading up to the excision of the primarytumor to evaluate therapies directed at inhibiting the early stages oftumor metastasis. Observations of morbidity and/or mortality arerecorded daily. Body weights are measured and recorded twice weekly.Potential endpoints include survival time, numbers of visible foci pertarget organ, or target organ weight. When survival time is used as theendpoint the other values are not determined. Survival data is used togenerate Kaplan-Meier curves. Significance is p≦0.05 by a log-rank testcompared to the control group in the experiment. The mean number ofvisible tumor foci as determined under a dissecting microscope, and themean target organ weights are compared by Student's t-test afterconducting an F-test, with significance determined at p≦0.05 compared tothe control group in the experiment for both of these endpoints.

4.2. Forced Metastasis

Tumor cells are injected into the tail vein, portal vein, or the leftventricle of the heart in experimental (forced) lung, liver, and bonemetastasis studies, respectively. Compounds are administered p.o., i.p.,i.v., i.m., or s.c. according to a predetermined schedule. Observationsof morbidity and/or mortality are recorded daily. Body weights aremeasured and recorded twice weekly. Potential endpoints include survivaltime, numbers of visible foci per target organ, or target organ weight.When survival time is used as the endpoint the other values are notdetermined. Survival data is used to generate Kaplan-Meier curves.Significance is p≦0.05 by a log-rank test compared to the control groupin the experiment. The mean number of visible tumor foci, as determinedunder a dissecting microscope, and the mean target organ weights arecompared by Student's t-test after conducting an F-test, withsignificance at p≦0.05 compared to the vehicle control group in theexperiment for both endpoints.

1-71. (canceled)
 72. An isolated and purified polynucleotide whichencodes an amino acid sequence selected from the group consisting of theamino acid sequences shown in SEQ ID NOS:2 and
 12. 73. Thepolynucleotide of claim 72 which comprises a nucleotide sequenceselected from the group consisting of the nucleotide sequences shown inSEQ ID NOS:1 and
 11. 74. The polynucleotide of claim 72 which is a cDNA.75. An isolated and purified single-stranded polynucleotide comprisingat least 1950 contiguous nucleotides of a coding sequence or acomplement of the coding sequence for an amino acid sequence selectedfrom the group consisting of the amino acid sequences shown in SEQ IDNOS:2 and
 12. 76. The polynucleotide of claim 75 wherein the proteincomprises an amino acid sequence selected from the group consisting ofthe amino acid sequences shown in SEQ ID NOS:2 and 12 and a nucleotidesequence selected from the group consisting of the nucleotide sequencesshown in SEQ ID NOS: 1 and
 11. 77. An expression construct, comprising;a coding sequence for an amino acid sequence selected from the groupconsisting of the amino acid sequences shown in SEQ ID NOS:2 and 12; anda promoter which is located upstream from the coding sequence and whichcontrols expression of the coding sequence.
 78. The expression constructof claim 77 wherein the coding sequence comprises a nucleotide sequenceselected from the group consisting of the nucleotide sequences shown inSEQ ID NOS:1 and
 11. 79. A host cell comprising an expression construct,wherein the expression construct comprises: a coding sequence for aprotein comprising an amino acid sequence selected from the groupconsisting of the amino acid sequences shown in SEQ ID NOS:2 and 12; anda promoter which is located upstream from the coding sequence and whichcontrols expression of the coding sequence.
 80. The host cell of claim79 which is prokaryotic.
 81. The host cell of claim 79 which iseukaryotic.
 82. A method of producing a protein, comprising the stepsof: culturing a host cell in a culture medium, wherein the host cellcomprises an expression construct comprising (a) a coding sequence for aprotein comprising an amino acid sequence selected from the groupconsisting of the amino acid sequences shown in SEQ ID NOS:2 and 12 and(b) a promoter which is located upstream from the coding sequence andwhich controls expression of the coding sequence, wherein the step ofculturing is carried out under conditions whereby the protein isexpressed; and recovering the protein.